I'm talking about ANCIENT china and japan.........Why did China and Japan move to self isolation from the international community???
The isolation allowed traditional art and writing to flourish and expand into more contemporary work untouched by western ideas. For two centuries during the Edo period in Japan the shogun refused western ships and missionaries entry to consolidate their power base and keep their customs pure. During this time Kabuki came about, just one of the indigenous explorations of creative expression.Why did China and Japan move to self isolation from the international community???
I think I learned somewhere sophomore year that they (China %26amp; Japan) started to realize that they were behind in technology (yes, I mean the technology during ancient times) and that it made them fall behind on what the other powerhouses (European states) could do and could gain more land for their empires. It also has to do with being able to keep up in their navies and weaponary, seeing that if they were still isolated, they could not trade with other people to obtain these supplies in order to make what they needed to gain land and power.. because back in the ancient times, certain places specialized in certain raw materials or refined materials because what was available to them on all the land that they owned. Also, there was no way to get certain items except by trade, and sea-trade was one of the ways to obtain these goods (land-trade is the other).
I hope that helps. I was trying to think of some more to tell you, but I couldn't exactly remember everything from AP World History two years ago. Haha, yeah.
Good luck. :)
power base== allows gov to est base over people...
complete loyalty...
both sought to control their domains and keep them in the way of life that were established. they were well aware of the greed of others, the need for conquest, the need to 'convert'. it was easier to just say no. we do not want that
Since their culture was drastically different then of Europeans they didn't want their cultures to assimilate and lose parts of their traditions.
Monday, August 23, 2010
What is animal quarantine? difference between quarantine and isolation HELLLLLP :(?
Im writing a report on it and dont understand, help pleasee. points given for best answer straight away if good xxx
What is animal quarantine? difference between quarantine and isolation HELLLLLP :(?
Quarantine is the holding of animals for a period of time until their disease status to some predetermine level of confidence is reached.
Isolation is putting animals in a separate room or building until used.
http://www.fauvet.fau.edu/oacm/Quarantin鈥?/a>
What is animal quarantine? difference between quarantine and isolation HELLLLLP :(?
Quarantine is a form of isolation. They isolate the animal long enough so if it has any diseases, there is time for the symtoms to present themselves. If the time period passes and so symptoms present, then the animal can leave. It is a way of ensuring that no exotic diseases are brought into the country.
Quarantining an animal is stricter than just isolating it. Records must be kept of who enters/exits the building/room it is being kept in, and no one is to disturb it.
Isolating an animal is just putting it off by itself, i.e. sorting out a calf from the herd and penning it because it looks sick. It's usually just to keep a common illness (like upper respiratory) from spreading among a group.
Because many diseases require time to incubate and show symptoms, quarantine would require separation from domestic stock (pets, etc.) during a period of careful observation for suspected disease, possibly including specific tests. Isolation may be required during quarantine but observations may not be required during isolation.
quarantine is when you can put a group of sick animals, or specific areas in isolation. isolation is when you strickly put one animal so to say in a confined area!! hope that answers your question
Can isolation be considered a theme?
i'm writing an essay on the Rime of the Ancient Mariner and Frankenstein so i was just wondering. ThanksCan isolation be considered a theme?
Isolation can be a great theme. I know you will write a great essay. If you need any help all you need to do is ask.Can isolation be considered a theme?
no, isolation is a topic.
I use this as reference when i'm confused:
Topic: Love
Theme: love conquers all.
Yes almost anything can be a theme.
Isolation can be a great theme. I know you will write a great essay. If you need any help all you need to do is ask.Can isolation be considered a theme?
no, isolation is a topic.
I use this as reference when i'm confused:
Topic: Love
Theme: love conquers all.
Yes almost anything can be a theme.
How did william golding's isolation impact on Lord of the flies?
by putting the british boys on an island
If the poor are isolated and the rich can afford isolation are they the same?
What are the differences?If the poor are isolated and the rich can afford isolation are they the same?
you force me to accept that the poor are isolated?
Nope, they live in PROJECTS, get free money and health care, free education and food...that's not ';isolated';
the rich pay MORE TAXES, pay for their own healthcare, home, food and education...they aren't ';isolated'; from SPENDING their money...If the poor are isolated and the rich can afford isolation are they the same?
Uh....sure. Why not?
One is a poor isolated person, while the other is a rich isolated person. Simple
No, they are polar opposites.
It is a symptom of a larger problem of the reversion to a class system of lords and serfs. The rich get richer and the poor get poorer, but the opportunity for the poor to excel is diminishing. It is slow, and insidious, but it is occuring. An example of this is the upcoming minimum wage increase. It will help to raise the quality of life for the lower class, but increase difficulties for many middle class because eventually, the cost of utilities and other living expense will rise to meet the new baseline living standard. So not only are the poor being isolated, they are being added to. In the meantime, the rich can sit back and allow this to happen as they take advantage of those who can't afford proper nutrition due to the cost or the time to attain it (thus resorting to McDonalds on a regular basis), work two jobs (thus not having time for proper health-maintenence), and poorer education.
The time is now. The chances to seize oppurtunity are dimishing and soon there will be no oppurtunity to be had. So therefore, those with power (i.e. the rich) will continue to isolate themselves and the poor to retain their power.
I don't think so: the poor are isolated because of economic limitations. They can't afford to experience the things the rich can do; can't afford to travel to exotic lands; can't afford to enjoy lavish dinners, luxury yachts, country club memberships, and all those other accoutrements that come with wealth.
The rich, on the other hand, can afford isolation because of unlimited economic resources. They can experience the things poor people can't do; they travel around the world; they enjoy lavish dinners, luxury yachts, country club memberships, and all those accoutrements that come with wealth. But they isolate themselves because they don't want to have to ';touched'; by the sights of the poor, disabled, homeless, unemployed, sick, aged, underprivileged, disadvantaged, or hungry. So they live in gated mansions, work in 'secured' office buildings, and pretty much stay away from anyone that's not in their economic or social class (I think it makes them feel guilty that they so much and those living in poverty have so little). -RKO-
Poor isolation is not voluntary while
Rich isolation is voluntary.
you force me to accept that the poor are isolated?
Nope, they live in PROJECTS, get free money and health care, free education and food...that's not ';isolated';
the rich pay MORE TAXES, pay for their own healthcare, home, food and education...they aren't ';isolated'; from SPENDING their money...If the poor are isolated and the rich can afford isolation are they the same?
Uh....sure. Why not?
One is a poor isolated person, while the other is a rich isolated person. Simple
No, they are polar opposites.
It is a symptom of a larger problem of the reversion to a class system of lords and serfs. The rich get richer and the poor get poorer, but the opportunity for the poor to excel is diminishing. It is slow, and insidious, but it is occuring. An example of this is the upcoming minimum wage increase. It will help to raise the quality of life for the lower class, but increase difficulties for many middle class because eventually, the cost of utilities and other living expense will rise to meet the new baseline living standard. So not only are the poor being isolated, they are being added to. In the meantime, the rich can sit back and allow this to happen as they take advantage of those who can't afford proper nutrition due to the cost or the time to attain it (thus resorting to McDonalds on a regular basis), work two jobs (thus not having time for proper health-maintenence), and poorer education.
The time is now. The chances to seize oppurtunity are dimishing and soon there will be no oppurtunity to be had. So therefore, those with power (i.e. the rich) will continue to isolate themselves and the poor to retain their power.
I don't think so: the poor are isolated because of economic limitations. They can't afford to experience the things the rich can do; can't afford to travel to exotic lands; can't afford to enjoy lavish dinners, luxury yachts, country club memberships, and all those other accoutrements that come with wealth.
The rich, on the other hand, can afford isolation because of unlimited economic resources. They can experience the things poor people can't do; they travel around the world; they enjoy lavish dinners, luxury yachts, country club memberships, and all those accoutrements that come with wealth. But they isolate themselves because they don't want to have to ';touched'; by the sights of the poor, disabled, homeless, unemployed, sick, aged, underprivileged, disadvantaged, or hungry. So they live in gated mansions, work in 'secured' office buildings, and pretty much stay away from anyone that's not in their economic or social class (I think it makes them feel guilty that they so much and those living in poverty have so little). -RKO-
Poor isolation is not voluntary while
Rich isolation is voluntary.
What is an Ultra isolation Transformer?
How to test / inspect an Ultra isolation Transformer?What is an Ultra isolation Transformer?
Usually these are made with two electrostatic shields between the primary windings and the secondary windings, and are connected to input ground for the first, and floating ground for the second.
Test it the way you would a normal transformer, and add a test for capacitance between secondary and primary windings, with the shields connected to the guard of the capacitance meter. You should read a very low capacitance. Then test each shield individually, then test capacitance between the shields.
.What is an Ultra isolation Transformer?
This is the first time I am hearing about such a device.
You take a look at www.howstuffworks.com
Usually these are made with two electrostatic shields between the primary windings and the secondary windings, and are connected to input ground for the first, and floating ground for the second.
Test it the way you would a normal transformer, and add a test for capacitance between secondary and primary windings, with the shields connected to the guard of the capacitance meter. You should read a very low capacitance. Then test each shield individually, then test capacitance between the shields.
.What is an Ultra isolation Transformer?
This is the first time I am hearing about such a device.
You take a look at www.howstuffworks.com
Why certain organisms,cells are more suitable for the isolation of high molecular weight DNA?
I would look at it the other way. Why is it hard to isolate high molecular weight DNA from certain cells.
If the they are bacteria, or some other single celled organism, then it could be an unusually tough cell wall that resists simple DNA extractions. If it is a multicelluar organism, but real small, well then you have all the other parts of the organism around, and again it might be a just a question of the exact method of DNA extraction. For particular cell types or tissues you could find that there are a lot of enzymes present that could degrade the DNA and unless the extraction technique takes that into account, you could end up with smaller DNA pieces.
In general the techniques need to be modified depending on the organism or tissue that is being used.eye make up
If the they are bacteria, or some other single celled organism, then it could be an unusually tough cell wall that resists simple DNA extractions. If it is a multicelluar organism, but real small, well then you have all the other parts of the organism around, and again it might be a just a question of the exact method of DNA extraction. For particular cell types or tissues you could find that there are a lot of enzymes present that could degrade the DNA and unless the extraction technique takes that into account, you could end up with smaller DNA pieces.
In general the techniques need to be modified depending on the organism or tissue that is being used.
In plasmid dna isolation why do we use new lysis solution every time?
lysis solution- NaOH and SDSIn plasmid dna isolation why do we use new lysis solution every time?
SDS degrades over time when in aqueous solution. Repeated freeze-thawing also doesn't help (if it's kept in cold storage).
SDS degrades over time when in aqueous solution. Repeated freeze-thawing also doesn't help (if it's kept in cold storage).
Which of the following provides for instant reproductive isolation?
a. polyploidy
b. most geographic barriers such as rivers and mountain belts
c. climate changes
d. behavior changes in females of the speciesWhich of the following provides for instant reproductive isolation?
If you can't physically get to your mate, its will make it hard for the two of you to reproduce...hope this helps :-D
b. most geographic barriers such as rivers and mountain belts
c. climate changes
d. behavior changes in females of the speciesWhich of the following provides for instant reproductive isolation?
If you can't physically get to your mate, its will make it hard for the two of you to reproduce...hope this helps :-D
What does geographic isolation lead to?
A. Ecological specialization
B. Adaptive radiation
C. Reproductive isolation
D. Increased species diversity
E. Sexual selectionWhat does geographic isolation lead to?
A.What does geographic isolation lead to?
I could see more than one of these answers being correct. Isolation may lead to ecological specialization, but not necessarily. I believe reproductive isolation is the best answer. Increased species diversity may be a possible outcome when viewing both the isolated and original populations as a whole, but diversity within the isolated group is likely to decrease.
C--Reproductive Isolation, which leads to speciation.
C and speciation
Australia - best example. So you figure it out form there.
A, C, or A and C. :)
B. Adaptive radiation
C. Reproductive isolation
D. Increased species diversity
E. Sexual selectionWhat does geographic isolation lead to?
A.What does geographic isolation lead to?
I could see more than one of these answers being correct. Isolation may lead to ecological specialization, but not necessarily. I believe reproductive isolation is the best answer. Increased species diversity may be a possible outcome when viewing both the isolated and original populations as a whole, but diversity within the isolated group is likely to decrease.
C--Reproductive Isolation, which leads to speciation.
C and speciation
Australia - best example. So you figure it out form there.
A, C, or A and C. :)
What is an example of speciation, geographic isolation and convergent evolution?
speciation - darwin's observation of the finches on the galopogos islands (please tell me i spelled that correctly)
geographic isolation - the polar bear and brown bear isolation due to the oceanic and glacier barriers
convergent evolution - the butterfly and the bird both have wings and use them for the same purpose, but they are not the same speciesWhat is an example of speciation, geographic isolation and convergent evolution?
speciation --%26gt; the finches on the galapagos islands
geographic isolation --%26gt; the finches on the galapagos islands
convergent --%26gt; two animals having an anagolous structure, like wings but from two different areas.
geographic isolation - the polar bear and brown bear isolation due to the oceanic and glacier barriers
convergent evolution - the butterfly and the bird both have wings and use them for the same purpose, but they are not the same speciesWhat is an example of speciation, geographic isolation and convergent evolution?
speciation --%26gt; the finches on the galapagos islands
geographic isolation --%26gt; the finches on the galapagos islands
convergent --%26gt; two animals having an anagolous structure, like wings but from two different areas.
In isolation of DNA why does phenol precipitate protein in the phenol/chloroform extraction?
because proteins are insoluble in phenol.
Extreme isolation experiences, what have you learned?
i have done 3 tours (isolation duty) in the military, and most of my jobs have been working alone but i usually come home to the dog (since the wife left) and read, or work on various projects.
in my time of isolation, i have learned a lot, about myself, my thinking and some that i have corresponded with by letters.
what have you learned?Extreme isolation experiences, what have you learned?
A long stint of seclusion taught me an awful lot about myself. Made me realize things I has pushed to the back of my memory, that I needed to address. And overall made me a healthier person, mentally.
Oddly enough, during that time I thought I'd go insane. Afterward I remember journaling that everyone should do it at least once. Extreme isolation experiences, what have you learned?
my friend you know that our perception is the only reality that we can ever know,so isolation is the real universe revealed. Report Abuse
A believer is not isolated from God (http://www.biblegateway.com/passage/?sea鈥?/a> ,) not that Jesus is God, but God is in Jesus.
I learn a lot too,it's good to learn about yourself. Be your own best friend also,know there are many others out there that have to be alone.
I'm never isolated. My kitten Oliver is always here and I end up talking to him and thinking to myself that I must be crazy.
LOL
I think we all could learn about ourself,s if we are alone
in our own solitude and stillness.
I have learned to meditate. How to still the mind and let whatever flow in without controlling it.
isolation is the reality.
How not to answer myself when I'm thinking out loud.eye make up
in my time of isolation, i have learned a lot, about myself, my thinking and some that i have corresponded with by letters.
what have you learned?Extreme isolation experiences, what have you learned?
A long stint of seclusion taught me an awful lot about myself. Made me realize things I has pushed to the back of my memory, that I needed to address. And overall made me a healthier person, mentally.
Oddly enough, during that time I thought I'd go insane. Afterward I remember journaling that everyone should do it at least once. Extreme isolation experiences, what have you learned?
my friend you know that our perception is the only reality that we can ever know,so isolation is the real universe revealed. Report Abuse
A believer is not isolated from God (http://www.biblegateway.com/passage/?sea鈥?/a> ,) not that Jesus is God, but God is in Jesus.
I learn a lot too,it's good to learn about yourself. Be your own best friend also,know there are many others out there that have to be alone.
I'm never isolated. My kitten Oliver is always here and I end up talking to him and thinking to myself that I must be crazy.
LOL
I think we all could learn about ourself,s if we are alone
in our own solitude and stillness.
I have learned to meditate. How to still the mind and let whatever flow in without controlling it.
isolation is the reality.
How not to answer myself when I'm thinking out loud.
What is meant by the statement,';Living things do not exist in isolation';?
It means that all living things need other living things in order to survive.What is meant by the statement,';Living things do not exist in isolation';?
I would guess that it means living things need other living things to survive such as a mammal cannot survive without oxygen, which is produced by plants. Therefore Mammals cannot live without plants.What is meant by the statement,';Living things do not exist in isolation';?
I think it means some what like this : Living beings cant live all alone,they must have some interaction with some1 or something, they cant live all alone and uneffected by any thing.
Nothing lives by itself. Most of the cells in a human body are not even human. There are the flora and fauna that live in our gut that help us digest our food. There are tiny mites that live in our hair folicles. This is true for all living things. The single cell organisms never live by themselves there are always others around.
I would guess that it means living things need other living things to survive such as a mammal cannot survive without oxygen, which is produced by plants. Therefore Mammals cannot live without plants.What is meant by the statement,';Living things do not exist in isolation';?
I think it means some what like this : Living beings cant live all alone,they must have some interaction with some1 or something, they cant live all alone and uneffected by any thing.
Nothing lives by itself. Most of the cells in a human body are not even human. There are the flora and fauna that live in our gut that help us digest our food. There are tiny mites that live in our hair folicles. This is true for all living things. The single cell organisms never live by themselves there are always others around.
My friend will be fine for weeks then go through an isolation week and not come out of his room...whats wrong?
My friend will be fine for weeks sometimes then something will happen and he will get PISSED at EVERYTHING and wont leave his room or about a week (not answering phone calls/front door...no contact what so ever with anyone) then after his episode he will be fine like nothing happened...wtf is up with that does he have like a disorder?My friend will be fine for weeks then go through an isolation week and not come out of his room...whats wrong?
i do the same exact thing. i suggest you research schzoid affective disorder, which is what i have. its a depression where you isolate yourself. an uncontrollable need to be alone. need your space. one minute you have to be around people but an overwhelming need takes over and you just need to be alone. people like us are able to keep ourselves busy and active better when alone. meds like celexa and abilify work for me.My friend will be fine for weeks then go through an isolation week and not come out of his room...whats wrong?
He might be suffering from depression or anxiety or a combination of things. Usually one can go days, week or months feeling fine but then something triggers and depression slips back in. The key signs are isolation and his mood. He needs to speak to a professional for a proper diagnoses.
And just try and be understanding during this time, he may need a little TLC...even if he acts like he doesn't.
I'm sorry to hear about your friend, but there could be a lot of underlying problems in his life, or possibly a mood disorder.
Depression or social anxiety will do this to you. These are caused by unbalanced seratonin (the horomone in your brain causing happiness/sometimes euphoric feelings) which means that it goes up and down at uncontrolled times causing the person to have sometimes very extensive mood swings. An anti-depressant could possibly help with the problem, but typically, depression is caused by a traumatic event or feelings that are low about themselves. A combination of therapy and an anti-depressant is a good start.
2nd, your friend could be bipolar. Some people are born with it, others are diagnosed later in life, again from traumatic events or low feelings and something happens to them that causes the ';manic'; state. Some people will go into huge outbursts for no reason, some even violent. Many don't sleep, isolate themselves, and become very disassociated with society itself. If this is the case, a psychotherapist/psychiatrist can diagnose the patient. Typically, a combination of medications sometimes with lithium will be prescribed. But please note, I am not a doctor by any means, and lithium levels must closely be monitored by a general practioner.
Good luck, I hope your friend is ok :)
Maybe Bipolar? I would try suggesting to him or his parents to get this checked out by a doctor if it's affecting his life.
Your friend might be an addict on a drug binge.
i do the same exact thing. i suggest you research schzoid affective disorder, which is what i have. its a depression where you isolate yourself. an uncontrollable need to be alone. need your space. one minute you have to be around people but an overwhelming need takes over and you just need to be alone. people like us are able to keep ourselves busy and active better when alone. meds like celexa and abilify work for me.My friend will be fine for weeks then go through an isolation week and not come out of his room...whats wrong?
He might be suffering from depression or anxiety or a combination of things. Usually one can go days, week or months feeling fine but then something triggers and depression slips back in. The key signs are isolation and his mood. He needs to speak to a professional for a proper diagnoses.
And just try and be understanding during this time, he may need a little TLC...even if he acts like he doesn't.
I'm sorry to hear about your friend, but there could be a lot of underlying problems in his life, or possibly a mood disorder.
Depression or social anxiety will do this to you. These are caused by unbalanced seratonin (the horomone in your brain causing happiness/sometimes euphoric feelings) which means that it goes up and down at uncontrolled times causing the person to have sometimes very extensive mood swings. An anti-depressant could possibly help with the problem, but typically, depression is caused by a traumatic event or feelings that are low about themselves. A combination of therapy and an anti-depressant is a good start.
2nd, your friend could be bipolar. Some people are born with it, others are diagnosed later in life, again from traumatic events or low feelings and something happens to them that causes the ';manic'; state. Some people will go into huge outbursts for no reason, some even violent. Many don't sleep, isolate themselves, and become very disassociated with society itself. If this is the case, a psychotherapist/psychiatrist can diagnose the patient. Typically, a combination of medications sometimes with lithium will be prescribed. But please note, I am not a doctor by any means, and lithium levels must closely be monitored by a general practioner.
Good luck, I hope your friend is ok :)
Maybe Bipolar? I would try suggesting to him or his parents to get this checked out by a doctor if it's affecting his life.
Your friend might be an addict on a drug binge.
What does the Dalie Lama do, and who finances him? I mean, The guy's been in isolation for 40 years.?
Most of these spiritual
';types'; come from wealth.
That is why they can give advice,
they don't have to worry about making money or the rat race.
Basically they are all full of it.What does the Dalie Lama do, and who finances him? I mean, The guy's been in isolation for 40 years.?
yes!
';types'; come from wealth.
That is why they can give advice,
they don't have to worry about making money or the rat race.
Basically they are all full of it.What does the Dalie Lama do, and who finances him? I mean, The guy's been in isolation for 40 years.?
yes!
Can years of social isolation drive a person insane and if so, how long will it take?
Hmm, some Buddhist Monks voluntarily isolate themselves for years in order to find a higher truth.
I think socialization is a bit like riding a bicycle- once you know how to interact with folks, you don't forget.Can years of social isolation drive a person insane and if so, how long will it take?
insane...? well that is relative.....social isolation pushes you inward, you only become insane to the outside world. Even though you become too unique for others to understand....your self-realization reaches new levels....this leads to ';insane'; behaviors that feel....natural :)....hope that makes sense...Can years of social isolation drive a person insane and if so, how long will it take?
it depends on the person, if you take advantage of the situation and do something useful you wouldn't go insane...but again everyone's different.
who says u need sumone to ASSESS the issue. ';voiceofreason'; is correct.i have been through that and experienced a higher understanding of life...
if he remains isolated there is no one with whom to interact ...so there is no one to assess the issue.
I think socialization is a bit like riding a bicycle- once you know how to interact with folks, you don't forget.Can years of social isolation drive a person insane and if so, how long will it take?
insane...? well that is relative.....social isolation pushes you inward, you only become insane to the outside world. Even though you become too unique for others to understand....your self-realization reaches new levels....this leads to ';insane'; behaviors that feel....natural :)....hope that makes sense...Can years of social isolation drive a person insane and if so, how long will it take?
it depends on the person, if you take advantage of the situation and do something useful you wouldn't go insane...but again everyone's different.
who says u need sumone to ASSESS the issue. ';voiceofreason'; is correct.i have been through that and experienced a higher understanding of life...
if he remains isolated there is no one with whom to interact ...so there is no one to assess the issue.
What is the best drawing to draw for isolation?
A single stone on a bare plain.What is the best drawing to draw for isolation?
draw a picture of any celebrity in a betty ford clinic.eye make up
draw a picture of any celebrity in a betty ford clinic.
Child kept in isolation for years?
I watched a documentary on psychology in the 20 century and how they used to remove part of the brains (Do you know what that is called?). And I remember one part they mentioned an event where a child was kept in one room for her whole life so couldn't walk,talk,etc. Do you anyone of you know of this event or where i can find more info?Child kept in isolation for years?
a lobotomy disconnects the frontal lobe.
feralchildren.com -----it has a huge amount of information on children raised by animals as well as kept isolated or in
cagesChild kept in isolation for years?
Well I found an example where something similar happened.http://www.learninginfo.org/genie-isolat鈥?/a>
Some children who are isolated from humans and live with another group animals are known as feral children.
This might be of help
http://www.feralchildren.com/en/children鈥?/a>
Wasn't that a documentary on tlc. Not sure where you could find information but I remember watching the same documentary.
a lobotomy disconnects the frontal lobe.
feralchildren.com -----it has a huge amount of information on children raised by animals as well as kept isolated or in
cagesChild kept in isolation for years?
Well I found an example where something similar happened.http://www.learninginfo.org/genie-isolat鈥?/a>
Some children who are isolated from humans and live with another group animals are known as feral children.
This might be of help
http://www.feralchildren.com/en/children鈥?/a>
Wasn't that a documentary on tlc. Not sure where you could find information but I remember watching the same documentary.
Which group experienced the most cultural isolation?
A.Maori
B. Hutus
C. TutsisWhich group experienced the most cultural isolation?
I'd say the Maori, they have much more geographic isolation than the other two and that always leads to a much more culturally isolated people.Which group experienced the most cultural isolation?
A. Maori
B
B
B. Hutus
C. TutsisWhich group experienced the most cultural isolation?
I'd say the Maori, they have much more geographic isolation than the other two and that always leads to a much more culturally isolated people.Which group experienced the most cultural isolation?
A. Maori
B
B
What is the function of the TE buffer in DNA isolation?
The isolation of DNA is one of the more commonly used procedures in many areas of bacterial physiology, genetics, molecular biology and biochemistry.
TE Buffer
10 mM Tris-Cl, pH 7.5 1 mM EDTA
TE buffer is often used to store DNA and RNA. The EDTA in TE chelates Mg2+ and other divalent metals ions necessary for most causes of DNA and RNA degradation, suppressing these processes. However, downstream reactions like restriction digests, PCR, ligations, and reverse transcription typically require Mg2+, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, you should keep track of the amount of EDTA in the mix to make sure there is still enough Mg2+ for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg2+ ion.
Some protocols use TE 10:0.1 with 0.1 mM EDTA to reduce the interaction of the EDTA with downstream applications.
Some people use TE buffers with different pH's for different applications. For example, DNA is stored at pH 8 to reduce depurination, which is acid catalyzed, while RNA is stored at a slightly lower pH (7.5) because degradation of RNA is base-catalyzed. Most downstream reactions will not be influenced by the slightly different pH storage conditions.
Retrieved from ';http://openwetware.org/wiki/TE_buffer';
http://www.waksmanfoundation.org/labs/wi鈥?/a>What is the function of the TE buffer in DNA isolation?
Buffer solution resists change in pH. It helps keep DNA's enviornment fairly neutral.
TE Buffer
10 mM Tris-Cl, pH 7.5 1 mM EDTA
TE buffer is often used to store DNA and RNA. The EDTA in TE chelates Mg2+ and other divalent metals ions necessary for most causes of DNA and RNA degradation, suppressing these processes. However, downstream reactions like restriction digests, PCR, ligations, and reverse transcription typically require Mg2+, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, you should keep track of the amount of EDTA in the mix to make sure there is still enough Mg2+ for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg2+ ion.
Some protocols use TE 10:0.1 with 0.1 mM EDTA to reduce the interaction of the EDTA with downstream applications.
Some people use TE buffers with different pH's for different applications. For example, DNA is stored at pH 8 to reduce depurination, which is acid catalyzed, while RNA is stored at a slightly lower pH (7.5) because degradation of RNA is base-catalyzed. Most downstream reactions will not be influenced by the slightly different pH storage conditions.
Retrieved from ';http://openwetware.org/wiki/TE_buffer';
http://www.waksmanfoundation.org/labs/wi鈥?/a>What is the function of the TE buffer in DNA isolation?
Buffer solution resists change in pH. It helps keep DNA's enviornment fairly neutral.
What is magnetic isolation?
I have a T6 PLC manufactured by Trol Systems. In the MCU-04-115 specs, the power requirements are 115VAC, .02A, 50/60 HZ w/1000v magnetic isolation. I understand everything except the w1000v magnetic isolation.
Over forty years playing with Electrical/Electronics I've never heard this term. We've used these for about 5 years and I just now noticed this. They work great and I don't have a problem with them. I'm just curious what they are telling me. Someone help me out.What is magnetic isolation?
Magnetic isolation is important sincereceivers produce undesired time varyingmagnetic fields outside their cases. Thesefields may be sensed by the hearing aidtelecoil and could create spurious inputsignals or cause a feedback loop. The VEFVibration Isolation receiver features twolevels of magnetic isolation. The case ofthe internal EF receiver acts at the primary magnetic shielding, or delicate wiring isrequired. The acoustic connection is madeat the port tube, which is rigid for easysealing. All delicate Litz wiring and wireloops needed for vibration isolation arealready completed inside the VEF receiver.Electrical connections are made by simplysoldering standard wire to the VEF terminalpads.Application GuidelinesThe VEF receiver suspension system ishighly integrated with the port tube andcase, resulting in a compact, vibration-reduced receiver system. This level ofmechanical integration and optimization,however, does place constraints on custommodifications for specific applications.Some changes that are normallystraightforward with traditional receiversmay be difficult to implement in the VEFVibration Isolation receiver.Coil changes are commonly requested andare straightforward to accomplish since theydo not affect the vibration isolation system.The same coil modifications that arepossible in EF receivers are also feasible inVEF receivers. Common changes includeimpedance changes, DC resistancechanges, two or three terminalconfigurations, and shock protected coils.The simplest approach involves reproducingthe electro-acoustic performance of anexisting EF receiver (e.g., EF-1937) in aVEF receiver (e.g., VEF-1937). Note thattwo terminal models may offer marginallybetter vibration isolation due to thesimplified internal wiring.
Over forty years playing with Electrical/Electronics I've never heard this term. We've used these for about 5 years and I just now noticed this. They work great and I don't have a problem with them. I'm just curious what they are telling me. Someone help me out.What is magnetic isolation?
Magnetic isolation is important sincereceivers produce undesired time varyingmagnetic fields outside their cases. Thesefields may be sensed by the hearing aidtelecoil and could create spurious inputsignals or cause a feedback loop. The VEFVibration Isolation receiver features twolevels of magnetic isolation. The case ofthe internal EF receiver acts at the primary magnetic shielding, or delicate wiring isrequired. The acoustic connection is madeat the port tube, which is rigid for easysealing. All delicate Litz wiring and wireloops needed for vibration isolation arealready completed inside the VEF receiver.Electrical connections are made by simplysoldering standard wire to the VEF terminalpads.Application GuidelinesThe VEF receiver suspension system ishighly integrated with the port tube andcase, resulting in a compact, vibration-reduced receiver system. This level ofmechanical integration and optimization,however, does place constraints on custommodifications for specific applications.Some changes that are normallystraightforward with traditional receiversmay be difficult to implement in the VEFVibration Isolation receiver.Coil changes are commonly requested andare straightforward to accomplish since theydo not affect the vibration isolation system.The same coil modifications that arepossible in EF receivers are also feasible inVEF receivers. Common changes includeimpedance changes, DC resistancechanges, two or three terminalconfigurations, and shock protected coils.The simplest approach involves reproducingthe electro-acoustic performance of anexisting EF receiver (e.g., EF-1937) in aVEF receiver (e.g., VEF-1937). Note thattwo terminal models may offer marginallybetter vibration isolation due to thesimplified internal wiring.
Is Isolation muscle have any benefits in Boxing?
Like Curls for bis, and leg ext....Is Isolation muscle have any benefits in Boxing?
I think compound movements such as squats, deadlifts, powerlifts, swinging heavy objects are more beneficial to gain the power you need to box.
For speed, exercises like plyometrics would help improve fast twitch muscles.eye make up
I think compound movements such as squats, deadlifts, powerlifts, swinging heavy objects are more beneficial to gain the power you need to box.
For speed, exercises like plyometrics would help improve fast twitch muscles.
Cooker Isolation switch and Legal Requirements?
I have just had a new kitchen installed with a dual fuel range cooker and the firm that have installed it have removed my old isolation switch for the old cooker and have not installed a new one. (My cooker was moved accross the room). Can anyone tell me if you are required by law to have an isolation switch?Cooker Isolation switch and Legal Requirements?
Yes you MUST have a means of isolation, and it should be within 2 meters of the cooker, if they moved it, did they not extend the existing cable. did you look inside the base unit cupboards, it may be located in there? The switch must have a high enough rating to switch the full load of the cooker. Make sure you get an electrical certificate for this work.Cooker Isolation switch and Legal Requirements?
Yes you are unless the supply is directly from the consumer unit and dedicated only for the cooker. If its dual fuel, it dosen't have to have the 45A contacts as per the old one. It needs to be able to cope with the maximum loading supplied to the cooker (I presume its the oven thats electric? - in which case a 13A isolator is usually adequate). The isolator must be visible and within 3 metres of the cooker itself.
They should have left you with a certificate to say the electrical work was carried out to standard and by an electrician who is registered to do such work. There are new regulations governing electrical installations in kitchens and bathrooms. I think you will find that you do need an isolation swithch for a cooker. We need one for an extractor fan.
As an ex contruction worker the answer is YES call the company back to install one or get another company to do it dont do it yourself it has to be a qualified electrician
Good answers!
Yes you MUST have a means of isolation, and it should be within 2 meters of the cooker, if they moved it, did they not extend the existing cable. did you look inside the base unit cupboards, it may be located in there? The switch must have a high enough rating to switch the full load of the cooker. Make sure you get an electrical certificate for this work.Cooker Isolation switch and Legal Requirements?
Yes you are unless the supply is directly from the consumer unit and dedicated only for the cooker. If its dual fuel, it dosen't have to have the 45A contacts as per the old one. It needs to be able to cope with the maximum loading supplied to the cooker (I presume its the oven thats electric? - in which case a 13A isolator is usually adequate). The isolator must be visible and within 3 metres of the cooker itself.
They should have left you with a certificate to say the electrical work was carried out to standard and by an electrician who is registered to do such work. There are new regulations governing electrical installations in kitchens and bathrooms. I think you will find that you do need an isolation swithch for a cooker. We need one for an extractor fan.
As an ex contruction worker the answer is YES call the company back to install one or get another company to do it dont do it yourself it has to be a qualified electrician
Good answers!
Is isolation a sign of depression?
Hi ALL,
I'm isolating myself recently. went through separation and tons of other changes in my life.
However, I am not sure I am depressed. for example I do not think I am sad most of the time. Is isolation a sign of depression or not necessarily. what other causes are there?
THANKS.Is isolation a sign of depression?
Yes, that's one of the classic signs of depression in fact. When I start to avoid people my family knows that it usually means that I'm going into a deep depression. That's when they call in professional help. Of course there are other signs of depression like over or under eating, sleeping more or less, not feeling enjoyment in things that you used to like to do. Wanting to avoid people places and things that you used to enjoy. If these symptoms last for more than 2 weeks you might be experincing a clinical depression.Is isolation a sign of depression?
You dont mention your age, but isolation is a problem if you are using it not to deal with whats bothering you. You are only as sick as your secrets! Find some one maybe a professional to talk to. There is nothing wrong with taking care of your mind as well as the rest of your body! Good Luck.
Not necessarily. I often distance myself from different groups of people, but I'm def. not depressed.
Isolation is a normal way for your body and mind to recover from a lot of changes at once. It just takes time to relax and get back on track.
Good luck!
yes, its the major symptom.the other symptoms are u might not feel asleep in nights,easily provoked n get angry for unecessary things.Not feel yr hunger,in some cases people may over eat etc.So better come out of this correct it at the wright time.
Yes.
I'm isolating myself recently. went through separation and tons of other changes in my life.
However, I am not sure I am depressed. for example I do not think I am sad most of the time. Is isolation a sign of depression or not necessarily. what other causes are there?
THANKS.Is isolation a sign of depression?
Yes, that's one of the classic signs of depression in fact. When I start to avoid people my family knows that it usually means that I'm going into a deep depression. That's when they call in professional help. Of course there are other signs of depression like over or under eating, sleeping more or less, not feeling enjoyment in things that you used to like to do. Wanting to avoid people places and things that you used to enjoy. If these symptoms last for more than 2 weeks you might be experincing a clinical depression.Is isolation a sign of depression?
You dont mention your age, but isolation is a problem if you are using it not to deal with whats bothering you. You are only as sick as your secrets! Find some one maybe a professional to talk to. There is nothing wrong with taking care of your mind as well as the rest of your body! Good Luck.
Not necessarily. I often distance myself from different groups of people, but I'm def. not depressed.
Isolation is a normal way for your body and mind to recover from a lot of changes at once. It just takes time to relax and get back on track.
Good luck!
yes, its the major symptom.the other symptoms are u might not feel asleep in nights,easily provoked n get angry for unecessary things.Not feel yr hunger,in some cases people may over eat etc.So better come out of this correct it at the wright time.
Yes.
Where ican get the report on -isolation of lactic acid bacteria to produce antimicrobial compound?
You should try PubMed. I entered your phrase and pulled up an article on goat's milk. See the link below.
You could try other websites as well. If you are in college, your college library should have links to various article and journal databases similar to PubMed.
You could try other websites as well. If you are in college, your college library should have links to various article and journal databases similar to PubMed.
Do you think economists focus on microeconomics and macroeconomics in isolation?
Absolutely not, most economists will have a small cat or song bird in a cage nearby that will keep them company while they run regressions. There is also the pizza guy who comes by on occasion to deliver energy providing sustenance. Alan Greenspan even married another human being, but most of us find the probability of that occurring again astronomical.
Do you think social isolation has an effect not only on elderly but young generation also.?
The youth of to day wants to spend time on the internet having impersonal contacts.making friends who ultimately remain, a mystery to them.Do u think that leads toward loneliness and depression.Personal communication between family members has diminished considerablyDo you think social isolation has an effect not only on elderly but young generation also.?
Funny you ask this question I'm currently reading a book called ';Social Intelligence'; Which touches on this subject as well as a few others plaguing our society today. The answer is yes humans are naturally a social species and IM is not even close to the same as real human contact. We have large areas of our brain devoted to interacting with others in our society. Each and every one of us has an effect on all of the people around us, even the ones we don't notice. Cells in our brain called mirror neurons react in accordance to the information that other people project at us.
Humans in isolation don't bode very well with the exception of very few. Being isolated for long times from other people can go as far as to cause delusions and hallucinations in people.Do you think social isolation has an effect not only on elderly but young generation also.?
It can do, yes of course and also stunt social interaction with peers and assimilation with cultures. The attitudes on the street are not necessarily those of peers on the internet. Also body language and intonation are abesent from online conversations, making the interpretation a little literal, methinks.
On the flipside, it can help shy people and the elderly/infirm by keeping them in touch with other people and events. So it does have it's benefits too (aint that what Psychology is all about, both sides of the coin?)
yes i do.....i think it has alot of impact on the young generation.
Are you talking about me? O.o It sounds like you're talking about me. Have you been spying on me?
This could be a yes or no thing, i believe it depends on the person. There are those who do better indirectly communicating with people and those who are social butterflies and they can balance there time between the computer and real life.
Those who in normal life dont do well talking to people have a place where they can communicate with people when they could have just locked themselves up in a room and throw away the key. This is their way of socializing, now that may not seen normal to most of us, but it helps them.
I think the key here is to find balance, humans do need others to talk to and to be with. Excluding yourself from social interactions can lead to mental illness in some cases or it could be a way for some not to fall into depression, i guess it just depends on the person in this situation. :)
You are so right. My grandson comes to visit and first thing he looks for is computer.
It is a stigma on our whole society now days.
The elderly don't choose their plight.
Uh...no. Not at all. If anything, it leads to a feeling of being loved and happiness. If one realized that he only communicated over the internet, that teen MAY feel depressed.
Social isolation is a problem at any age. I think the internet is another tool to be used to communicate with each other, and so a very good thing. Time spent learning, reading, communicating with colleagues, friends, distant family, or new people you meet. But for those of us on the computer a lot, interacting with people on line doesn't mean you can't take the time for the people in your house.
It is definitely changing things for the kids. But remember the old system had severe problems, it was nearly impossible for introverted kids to be popular, and the need to belong to a group was responsible for much evil in our society. Much of the drive to be popular ended up using drugs and doing self-destructive things. It might be an improvement to do things with the Internet, Yahoo answers does produce some wisdom now and then, and that is better than the school yard popular contests.
Absolutely. It's sad.
I think it would need to be a case by case basis. It can have those effects. Dependant on the maturity of the child. Also in the home, the family need to limit a childs time on the computer and encourage interaction in the home with family.
Most kids attend school, church and other social activities as well as the home internet time. If a child is being allowed to not attend school, and allowed to spend the entire day on the internet and computer games, then issues in the home need to be changed. I feel it often still comes down to parental responsiblity to monitor and set limits.
I am from this present generation and i strongly agree with you.
I am a very social person and like to make friends and interact with people.
But, i am new to the US and buffered by the local people I want to meet an interact and talk. And the people interested in talking to me always seem to have some hidden agenda (religious or other) and I often tend to turn to the internet to make friends, but it sometimes lacks the personal touch and makes you feel lonely and depressed. I also happen to miss my family a lot , so i guess that factors in too.
You just consciously seek to be accepted and hope someone out there can cuddle you and make you feel warm inside.
isolation is good these days with all the freaky crazy people out there your safer in then out
It can do, yes of course and also stunt social interaction with peers and assimilation with cultures. The attitudes on the street are not necessarily those of peers on the internet. Also body language and intonation are abesent from online conversations, making the interpretation a little literal, methinks.
On the flipside, it can help shy people and the elderly/infirm by keeping them in touch with other people and events. So it does have it's benefits too (aint that what Psychology is all about, both sides of the coin?)
Truly this is a blessing and a curse. On the one hand the greatest information stop in the world and in the other we hypnotism all but. I say yes because to much of anything does make you an addict.
Depression and loneliness these are some characteristic of an addict.
I'm a father of three who is very active,because I have three sons. I spend time with them on the video game but I also make them get off and get outside sometimes they may mean I have to do a couple kick flips on my board for them to go out, but whatever helps them to keep realizing that there's a whole other world out there and to utilize it.
The need is that they want someone to talk to be that someone show interest in what there doing don't get me wrong theres a level of privacy but just show the interest.
Funny you ask this question I'm currently reading a book called ';Social Intelligence'; Which touches on this subject as well as a few others plaguing our society today. The answer is yes humans are naturally a social species and IM is not even close to the same as real human contact. We have large areas of our brain devoted to interacting with others in our society. Each and every one of us has an effect on all of the people around us, even the ones we don't notice. Cells in our brain called mirror neurons react in accordance to the information that other people project at us.
Humans in isolation don't bode very well with the exception of very few. Being isolated for long times from other people can go as far as to cause delusions and hallucinations in people.Do you think social isolation has an effect not only on elderly but young generation also.?
It can do, yes of course and also stunt social interaction with peers and assimilation with cultures. The attitudes on the street are not necessarily those of peers on the internet. Also body language and intonation are abesent from online conversations, making the interpretation a little literal, methinks.
On the flipside, it can help shy people and the elderly/infirm by keeping them in touch with other people and events. So it does have it's benefits too (aint that what Psychology is all about, both sides of the coin?)
yes i do.....i think it has alot of impact on the young generation.
Are you talking about me? O.o It sounds like you're talking about me. Have you been spying on me?
This could be a yes or no thing, i believe it depends on the person. There are those who do better indirectly communicating with people and those who are social butterflies and they can balance there time between the computer and real life.
Those who in normal life dont do well talking to people have a place where they can communicate with people when they could have just locked themselves up in a room and throw away the key. This is their way of socializing, now that may not seen normal to most of us, but it helps them.
I think the key here is to find balance, humans do need others to talk to and to be with. Excluding yourself from social interactions can lead to mental illness in some cases or it could be a way for some not to fall into depression, i guess it just depends on the person in this situation. :)
You are so right. My grandson comes to visit and first thing he looks for is computer.
It is a stigma on our whole society now days.
The elderly don't choose their plight.
Uh...no. Not at all. If anything, it leads to a feeling of being loved and happiness. If one realized that he only communicated over the internet, that teen MAY feel depressed.
Social isolation is a problem at any age. I think the internet is another tool to be used to communicate with each other, and so a very good thing. Time spent learning, reading, communicating with colleagues, friends, distant family, or new people you meet. But for those of us on the computer a lot, interacting with people on line doesn't mean you can't take the time for the people in your house.
It is definitely changing things for the kids. But remember the old system had severe problems, it was nearly impossible for introverted kids to be popular, and the need to belong to a group was responsible for much evil in our society. Much of the drive to be popular ended up using drugs and doing self-destructive things. It might be an improvement to do things with the Internet, Yahoo answers does produce some wisdom now and then, and that is better than the school yard popular contests.
Absolutely. It's sad.
I think it would need to be a case by case basis. It can have those effects. Dependant on the maturity of the child. Also in the home, the family need to limit a childs time on the computer and encourage interaction in the home with family.
Most kids attend school, church and other social activities as well as the home internet time. If a child is being allowed to not attend school, and allowed to spend the entire day on the internet and computer games, then issues in the home need to be changed. I feel it often still comes down to parental responsiblity to monitor and set limits.
I am from this present generation and i strongly agree with you.
I am a very social person and like to make friends and interact with people.
But, i am new to the US and buffered by the local people I want to meet an interact and talk. And the people interested in talking to me always seem to have some hidden agenda (religious or other) and I often tend to turn to the internet to make friends, but it sometimes lacks the personal touch and makes you feel lonely and depressed. I also happen to miss my family a lot , so i guess that factors in too.
You just consciously seek to be accepted and hope someone out there can cuddle you and make you feel warm inside.
isolation is good these days with all the freaky crazy people out there your safer in then out
It can do, yes of course and also stunt social interaction with peers and assimilation with cultures. The attitudes on the street are not necessarily those of peers on the internet. Also body language and intonation are abesent from online conversations, making the interpretation a little literal, methinks.
On the flipside, it can help shy people and the elderly/infirm by keeping them in touch with other people and events. So it does have it's benefits too (aint that what Psychology is all about, both sides of the coin?)
Truly this is a blessing and a curse. On the one hand the greatest information stop in the world and in the other we hypnotism all but. I say yes because to much of anything does make you an addict.
Depression and loneliness these are some characteristic of an addict.
I'm a father of three who is very active,because I have three sons. I spend time with them on the video game but I also make them get off and get outside sometimes they may mean I have to do a couple kick flips on my board for them to go out, but whatever helps them to keep realizing that there's a whole other world out there and to utilize it.
The need is that they want someone to talk to be that someone show interest in what there doing don't get me wrong theres a level of privacy but just show the interest.
What are the advantages and disadvantages of China's geographic isolation?
advantages
secure from monsoons by the mountains
when china first inhabited, good for safety from enemies
disadvantages
difficult for communicationeye make up
secure from monsoons by the mountains
when china first inhabited, good for safety from enemies
disadvantages
difficult for communication
How come, after RNA isolation, PCR shows two bands (18S 28S) but these bands dont show after DNase I treat?
RNA is pretty delicate stuff- most likely your kit is degrading it. I was using a kit that involved heating the RNA following DNase treatment to 70 degrees and this completely degraded the RNA as seen on the agilent bioanalyser. My advice would be to test the quality using the agilent/experion bioanalyser for 18 and 28s. Also look for RNA isolation kits or individual DNase kits that involve eluting off columns/ gel matrix and these work fine for me!How come, after RNA isolation, PCR shows two bands (18S 28S) but these bands dont show after DNase I treat?
May be it got chewed up into tiny pieces and ran off your gel.How come, after RNA isolation, PCR shows two bands (18S 28S) but these bands dont show after DNase I treat?
are you using a kit? a similar thing happened to us, and we just switched companies and the samples worked fine
It has been my experience that some DNAse I preparations are contaminated with RNAse! Try to obtain some RNAse free DNAse and try again.
Good luck.
Your question is nonsense. Please give more details. Like did you do a reverse transcription reaction on the RNA first. What primer set did you use for the PCR etc.
If you got a PCR product before DNAse treatment, but not afterwards then your RNA was contaminated with DNA and your PCR was probably just amplifying genomic DNA. Anyway please clarify what you did.
May be it got chewed up into tiny pieces and ran off your gel.How come, after RNA isolation, PCR shows two bands (18S 28S) but these bands dont show after DNase I treat?
are you using a kit? a similar thing happened to us, and we just switched companies and the samples worked fine
It has been my experience that some DNAse I preparations are contaminated with RNAse! Try to obtain some RNAse free DNAse and try again.
Good luck.
Your question is nonsense. Please give more details. Like did you do a reverse transcription reaction on the RNA first. What primer set did you use for the PCR etc.
If you got a PCR product before DNAse treatment, but not afterwards then your RNA was contaminated with DNA and your PCR was probably just amplifying genomic DNA. Anyway please clarify what you did.
Does anyone else feel a real strong need for isolation?
This is a lifetime thing with me and even though I have a lot of longtime friends and happily married there are times I feel Iwill 'loose it' if I can't go somewhere and be alone for at least a day.Does anyone else feel a real strong need for isolation?
its called a mental health break more people should try it.Does anyone else feel a real strong need for isolation?
Well, I am happy when I'm with my friends, but I definitely require ';me'; time, too. Being alone does not bother me at all.
Everyone needs private space and time. TAKE it. Most people that care about you will understand and applaud your honesty.
Yep! I need a fair amount of alone time. Time to let things run in my head. I need a couple hours a day. I garden so during my gardening time I get the quiet respite I need. I had my first week off of work in two years and I went home and unplugged my phone for one week and parked my car at the very end of my drive way so no one could easily stop by for a visit. Some people require absolutely NO alone time whatsoever, I am not one of them!!!
I think the more you think, the more alone time you need.
I think most people need some ';private time';. We live in a busy world and marriages today are very hectic with everyone working and trying to raise families. In order to get some private time for myself, I made an extra bedroom into an ';office'; so that when needed, I can go in and close the door. Sometimes I have to ask others to pardon me for a while but this never seems to be a problem.
No, there is a strong need for good friends and family connections.
its called a mental health break more people should try it.Does anyone else feel a real strong need for isolation?
Well, I am happy when I'm with my friends, but I definitely require ';me'; time, too. Being alone does not bother me at all.
Everyone needs private space and time. TAKE it. Most people that care about you will understand and applaud your honesty.
Yep! I need a fair amount of alone time. Time to let things run in my head. I need a couple hours a day. I garden so during my gardening time I get the quiet respite I need. I had my first week off of work in two years and I went home and unplugged my phone for one week and parked my car at the very end of my drive way so no one could easily stop by for a visit. Some people require absolutely NO alone time whatsoever, I am not one of them!!!
I think the more you think, the more alone time you need.
I think most people need some ';private time';. We live in a busy world and marriages today are very hectic with everyone working and trying to raise families. In order to get some private time for myself, I made an extra bedroom into an ';office'; so that when needed, I can go in and close the door. Sometimes I have to ask others to pardon me for a while but this never seems to be a problem.
No, there is a strong need for good friends and family connections.
If you were condemned to live in TOTAL ISOLATION, in constant darkness, for the rest of your LIFE...?
....with water and food delivered daily to keep you alive, would you be able to make the most of the adverse circumstances and try to create a world of your own where life was tolerable and enjoyable, regardless?If you were condemned to live in TOTAL ISOLATION, in constant darkness, for the rest of your LIFE...?
if you were in that place since you were born, you would have no basis as to what a ';tolerable'; life is. So theoretically, you can enjoy it. (based on your persepective). But when viewed from our point, that is not really ';enjoyable';.
but if you were living a normal life and then you've been dragged to that condition, it would be very agonizing because your brain will compare your previous and present conditions. you may try to meditate/concentrate(or be accustomed to it) to make it more tolerable. (but given a choice, you will still prefer being free from the situation.)
i hope your physics teacher taught you about reference frames. have a nice day.If you were condemned to live in TOTAL ISOLATION, in constant darkness, for the rest of your LIFE...?
I would try, probably unsuccessfully, but try nevertheless.
A bit of meditation =)
acknowledging the partially insane part of me i would probably make up friends or just live inside my head, dreams, stories, and etc.
No. I would spend each waking moment thinking about everything i used to have.how i took it for granted.sinking in a sea of despair.....i would kill myself.
if you were in that place since you were born, you would have no basis as to what a ';tolerable'; life is. So theoretically, you can enjoy it. (based on your persepective). But when viewed from our point, that is not really ';enjoyable';.
but if you were living a normal life and then you've been dragged to that condition, it would be very agonizing because your brain will compare your previous and present conditions. you may try to meditate/concentrate(or be accustomed to it) to make it more tolerable. (but given a choice, you will still prefer being free from the situation.)
i hope your physics teacher taught you about reference frames. have a nice day.If you were condemned to live in TOTAL ISOLATION, in constant darkness, for the rest of your LIFE...?
I would try, probably unsuccessfully, but try nevertheless.
A bit of meditation =)
acknowledging the partially insane part of me i would probably make up friends or just live inside my head, dreams, stories, and etc.
No. I would spend each waking moment thinking about everything i used to have.how i took it for granted.sinking in a sea of despair.....i would kill myself.
Where can I purchase a ZDRDCT10-4 EMI Isolation Tap for cable TV without going to the cable company?
Best Buy -or- Circuit City!Where can I purchase a ZDRDCT10-4 EMI Isolation Tap for cable TV without going to the cable company?
http://cu1.stores.yahoo.net/chanviscvt15鈥?/a>
is probably what you're looking for
http://cu1.stores.yahoo.net/chanviscvt15鈥?/a>
is probably what you're looking for
What does geographic isolation lead to?
Incestual tendencies and hemopheliaWhat does geographic isolation lead to?
Biologically, it leads to speciation.
Culturally, it leads to stasis.What does geographic isolation lead to?
Geographic isolation leads to either stagnation (biologically, economically, socially, and technologically) or extermination, and often both.
For starters, a group existing in geographic isolation will either adapt to it or die off. Provided they are able to adapt and they they will more or less achieve an equillibrium with their environment where they do what is necessary to survive and live reasonably comfortably. Once they have achieved this equilibrium however they will stagnate because there is no impetus for further development and adaptation.
This represents a serious danger to the group however should their situation change. If there is a dramatic climate/conditions change then they will be faced once again with adaptation of extermination, often with unpleasant consequences.
For reference, see the dinosaurs, perfectly adapted to their world and then their world changed and out they went.
Another danger is the end of that isolation. Because the group has existed in isolation they have biologically stagnated, particularly in regards to its resistance to diseases. As such, when new microbes are introduced they will have little resistence too it and thus be exterminated.
For reference to this, see the mass extermination of native Americans in both north AND south America following contact with Eurasians. The same also applies to the stone age tribes in new guinea and throughout the pacific islands, australia, and even the iron age tribes in Africa.
These consequences are as true for cultural norms as they are for biological ones. Culture in this case encompassing economic, social, and technological norms. History shows, time and again, that groups that exist within a bubble come out far worse off once that bubble is broken.
For reference... just look at world history from the ancient to the modern. It is EXTREMELY rare for cultures that exist in isolation to come off the better once that isolation is broken. Even the most successful, Japan, was still harsh and resulted in a total reworking of their culture so that it bears little more than surface resemblance to its ancient roots.
Inbreeding.
Biologically, it leads to speciation.
Culturally, it leads to stasis.What does geographic isolation lead to?
Geographic isolation leads to either stagnation (biologically, economically, socially, and technologically) or extermination, and often both.
For starters, a group existing in geographic isolation will either adapt to it or die off. Provided they are able to adapt and they they will more or less achieve an equillibrium with their environment where they do what is necessary to survive and live reasonably comfortably. Once they have achieved this equilibrium however they will stagnate because there is no impetus for further development and adaptation.
This represents a serious danger to the group however should their situation change. If there is a dramatic climate/conditions change then they will be faced once again with adaptation of extermination, often with unpleasant consequences.
For reference, see the dinosaurs, perfectly adapted to their world and then their world changed and out they went.
Another danger is the end of that isolation. Because the group has existed in isolation they have biologically stagnated, particularly in regards to its resistance to diseases. As such, when new microbes are introduced they will have little resistence too it and thus be exterminated.
For reference to this, see the mass extermination of native Americans in both north AND south America following contact with Eurasians. The same also applies to the stone age tribes in new guinea and throughout the pacific islands, australia, and even the iron age tribes in Africa.
These consequences are as true for cultural norms as they are for biological ones. Culture in this case encompassing economic, social, and technological norms. History shows, time and again, that groups that exist within a bubble come out far worse off once that bubble is broken.
For reference... just look at world history from the ancient to the modern. It is EXTREMELY rare for cultures that exist in isolation to come off the better once that isolation is broken. Even the most successful, Japan, was still harsh and resulted in a total reworking of their culture so that it bears little more than surface resemblance to its ancient roots.
Inbreeding.
';Bin cages'; for a hermit crab's isolation?
I have a plasic bin that is a little bigger then the critter carriers. Would it be okay if I got a heating pad, drilled holes in the top, and used it for an isolation tank for hermie molting?';Bin cages'; for a hermit crab's isolation?
for a moulting container no because plastic cannot be heated to the levels it needs to and it's harder to keep the humidity up.
but for mites it would work.
I stopped isolating healthy moulters a few years ago no issues yet.eye make up
for a moulting container no because plastic cannot be heated to the levels it needs to and it's harder to keep the humidity up.
but for mites it would work.
I stopped isolating healthy moulters a few years ago no issues yet.
How does isolation hurt a person?
people who willingly isolate themselves...they live in a very small world, they dont have close friends or dont want anyone close to them and instead spend time reading or i dont know dreaming maybe...and they are asexual...
what can be done a save a person like this?How does isolation hurt a person?
It can make them depressed, frustrated, because they are not interacting with people, they can see the world go by outside their window, on the TV, people interacting with each other, yet they hold themselves away from it all.
They can start to question things more because they are isolated, start having fantasies, long term isolation can deeply affect a personality and how they view people, if the person is young, isolation away from people, can decrease their sense of humor, the person will not fit in with others of the same age, but rather have a more serious perspective of the world, can think like a pessimist.How does isolation hurt a person?
Well some people might isolate themselves because they feel the world will not understand them. Maybe make them feel like their not alone and that people actually understand. Plus if the persons isolated like I said it could make them feel like that only they understand themselves and no one else, in some cases of love the couple might feel wronged by the ignorant world we live in so they isolate themselves from everyone else. Could or might lead to depression.
it depends on why they isolate themselves, and how isolated they are.
you said they live in a very small world, they don't have close friends.
that is not necessarily true. I live in a very rural area, don't see people often except my immediate family, the reason we live like this is, we do not want to be bothered by drunken louts, drug addicts, general dog and litter mess all over the pavements. we like to be able to breathe fresh air, enjoy reading, like walking and fishing. it does not mean we are isolating ourselves from everyone, it does not mean we do not have close friends, just that our friends are that close they understand we do not have to live close to be close and as such respect us as we respect them.
I am comfortable in crowds, I can go out with friends and have a laugh, I just do not do it frequently.
It does nothing to my psyche being isolated, it does give me a lot more time to think and be patient than what I normally find from people who never give themselves any quite time.
if you are meaning hermits, then maybe they are fed up with people, society, the way life has become, then they don't want saved by going into a town, they want to be free and live by a standard they feel comfortable with.
not everyone who lives in isolation is a fruitcake.
what can be done a save a person like this?How does isolation hurt a person?
It can make them depressed, frustrated, because they are not interacting with people, they can see the world go by outside their window, on the TV, people interacting with each other, yet they hold themselves away from it all.
They can start to question things more because they are isolated, start having fantasies, long term isolation can deeply affect a personality and how they view people, if the person is young, isolation away from people, can decrease their sense of humor, the person will not fit in with others of the same age, but rather have a more serious perspective of the world, can think like a pessimist.How does isolation hurt a person?
Well some people might isolate themselves because they feel the world will not understand them. Maybe make them feel like their not alone and that people actually understand. Plus if the persons isolated like I said it could make them feel like that only they understand themselves and no one else, in some cases of love the couple might feel wronged by the ignorant world we live in so they isolate themselves from everyone else. Could or might lead to depression.
it depends on why they isolate themselves, and how isolated they are.
you said they live in a very small world, they don't have close friends.
that is not necessarily true. I live in a very rural area, don't see people often except my immediate family, the reason we live like this is, we do not want to be bothered by drunken louts, drug addicts, general dog and litter mess all over the pavements. we like to be able to breathe fresh air, enjoy reading, like walking and fishing. it does not mean we are isolating ourselves from everyone, it does not mean we do not have close friends, just that our friends are that close they understand we do not have to live close to be close and as such respect us as we respect them.
I am comfortable in crowds, I can go out with friends and have a laugh, I just do not do it frequently.
It does nothing to my psyche being isolated, it does give me a lot more time to think and be patient than what I normally find from people who never give themselves any quite time.
if you are meaning hermits, then maybe they are fed up with people, society, the way life has become, then they don't want saved by going into a town, they want to be free and live by a standard they feel comfortable with.
not everyone who lives in isolation is a fruitcake.
Prison isolation - cruel and unusual punishment?
I'm just wondering if putting immates ';in the hole';, as it used to be termed, ie in isolation for being especially hostile, repeatedly breaking prison rules, etc, was considered cruel and unusual punishment. I seem to recall reading about a case in the '70s where it was determined (in Arkansas, I believe it was) that any period longer than 30 days constituted C%26amp;U, but I haven't been able to find any info besides that.Prison isolation - cruel and unusual punishment?
I read that same case somewhere. Whereas 29 days isn鈥檛 CU but 30 is? It goes along with a lot of criminal determinations which are based on time. Before I get into that I will answer your original question.
23 hours in solitary today I believe is not considered CU. A prisoner gets one hour of day light per day.
Now, 29 days = Not; but 30 = Yes.
One of the things I have seen with some current criminal larceny statutes is that with rental agreements they create a presumptive intent after X # of days. For example, you rent a move and hold it for 9 days = OK. If you hold it for 10 days the code creates a presumption of guilt when the clock strikes that 10th day. With that presumption and Intent being an element of larceny it has shifted the burden on to the defense on that 10th day. Meaning a dead person could manifest intent.
I read that same case somewhere. Whereas 29 days isn鈥檛 CU but 30 is? It goes along with a lot of criminal determinations which are based on time. Before I get into that I will answer your original question.
23 hours in solitary today I believe is not considered CU. A prisoner gets one hour of day light per day.
Now, 29 days = Not; but 30 = Yes.
One of the things I have seen with some current criminal larceny statutes is that with rental agreements they create a presumptive intent after X # of days. For example, you rent a move and hold it for 9 days = OK. If you hold it for 10 days the code creates a presumption of guilt when the clock strikes that 10th day. With that presumption and Intent being an element of larceny it has shifted the burden on to the defense on that 10th day. Meaning a dead person could manifest intent.
Give an example of an animal that became a species because of geographic isolation?
Darwin's finches.Give an example of an animal that became a species because of geographic isolation?
Just about anything in Australia.
Just about anything in Australia.
Saturday, August 21, 2010
Would you like to live in isolation, such as an island or a moor?
Or do you prefer the city and need people around you? I personally would love a slow pace of life without clock watching all the time.Would you like to live in isolation, such as an island or a moor?
i think they call that heaven on earth lol yes i would love it! i dont think any one wold choose to live in the busling city with all the crime and smog noway you definitly have the best ideaWould you like to live in isolation, such as an island or a moor?
i like silence,i have a watch but i don't look at the hour neither
is a stress giver tool
sure just be a bum.
Humans, being highly social animals, have a strong inherrent desire to live communally!
Though the whole idea of 'getting away from the crowd' is a nice thought - in reality, and over a period of time, you would begin to miss the company of others.
In a city, at least, you DO have an opportunity to cut yourself away from the often madding crowd by locking yourself in the bathroom! You have the options. On a moor - you have but one option: To mix it with the sheep or be lonely!
I have proven to achive peace in both places. I prefer many near me living never wanting to isolate or sharing and being available to others,and them being available to me! how wonderful to have the mass of support!
I quite fancy the idea of a lighthouse. But I would have to have access to the shops occasionally!
Lately I have been feeling that way, but it's just my moodswings again.
When the thought crosses my mind it does sound intersting for maybe two weeks to be on a Island with plenty of Beer.
I am an isolationist. I am in the process of building a cabin in the middle of my private woodland where I intenet to live away from the hustle and bustle of town life.
MY TYPE OF A PERSON ........................
I like the city it's what i'm used to, if it's too quiet i'll have to put some music on, i've the advantage of just being an hours drive from peace and quiet when it need though.
i think they call that heaven on earth lol yes i would love it! i dont think any one wold choose to live in the busling city with all the crime and smog noway you definitly have the best ideaWould you like to live in isolation, such as an island or a moor?
i like silence,i have a watch but i don't look at the hour neither
is a stress giver tool
sure just be a bum.
Humans, being highly social animals, have a strong inherrent desire to live communally!
Though the whole idea of 'getting away from the crowd' is a nice thought - in reality, and over a period of time, you would begin to miss the company of others.
In a city, at least, you DO have an opportunity to cut yourself away from the often madding crowd by locking yourself in the bathroom! You have the options. On a moor - you have but one option: To mix it with the sheep or be lonely!
I have proven to achive peace in both places. I prefer many near me living never wanting to isolate or sharing and being available to others,and them being available to me! how wonderful to have the mass of support!
I quite fancy the idea of a lighthouse. But I would have to have access to the shops occasionally!
Lately I have been feeling that way, but it's just my moodswings again.
When the thought crosses my mind it does sound intersting for maybe two weeks to be on a Island with plenty of Beer.
I am an isolationist. I am in the process of building a cabin in the middle of my private woodland where I intenet to live away from the hustle and bustle of town life.
MY TYPE OF A PERSON ........................
I like the city it's what i'm used to, if it's too quiet i'll have to put some music on, i've the advantage of just being an hours drive from peace and quiet when it need though.
Electrolysis of molten MgCl2 is the final production step in the isolation of magnesium from seawater by ....?
Electrolysis of molten MgCl2 is the final production step in the isolation of magnesium from seawater by the Dow process. When 34.4 g Mg metal forms, calculate the following.
(a) How many moles of electrons are required?
(b) How many coulombs are required?
(c) How many amps are required to produce this amount in 3.00 h?
please help.Electrolysis of molten MgCl2 is the final production step in the isolation of magnesium from seawater by ....?
Molar mass of Mg = 24.31 g/mol
Mole of Mg produced = 34.4 g / 24.31 g/mol = 1.415 mol
(a) Reduction equation;
Mg^2+(aq) + 2e- ------%26gt; Mg(s)
For each mole of Mg produced, 2 mole of e- are required.
For 1.415 mole of Mg; 2 x 1.415 = 2.83 mol e- are required.
(b) The charge of 1 mol e- is 1 F which is 96485 coulombs,
The charge of 2.83 mol e- ;
2.83 mol e- x 96485 coulombs / mol e- = 273,053 coulombs.
(c) Ampere = Coulombs / second
Time = 3.00 h x 3600 sec / hr = 10800 sec
Ampere = 273053 / 10800 = 25.28 A
(a) How many moles of electrons are required?
(b) How many coulombs are required?
(c) How many amps are required to produce this amount in 3.00 h?
please help.Electrolysis of molten MgCl2 is the final production step in the isolation of magnesium from seawater by ....?
Molar mass of Mg = 24.31 g/mol
Mole of Mg produced = 34.4 g / 24.31 g/mol = 1.415 mol
(a) Reduction equation;
Mg^2+(aq) + 2e- ------%26gt; Mg(s)
For each mole of Mg produced, 2 mole of e- are required.
For 1.415 mole of Mg; 2 x 1.415 = 2.83 mol e- are required.
(b) The charge of 1 mol e- is 1 F which is 96485 coulombs,
The charge of 2.83 mol e- ;
2.83 mol e- x 96485 coulombs / mol e- = 273,053 coulombs.
(c) Ampere = Coulombs / second
Time = 3.00 h x 3600 sec / hr = 10800 sec
Ampere = 273053 / 10800 = 25.28 A
When innoculating agar plate, Is streaking the plate same as isolation technique? Please clarify.?
Cultures ordered from a supply company or stock center will probably not consist of genetically identical bacteria. The bacteria will all be of the same species, and available as a single strain. However, random mutations may still exist due to the large number of bacteria present. To obtain a source of genetically identical bacteria, streak plates are used. Streaking a plate allows the bacteria to be spread out so that a single bacterium can be isolated from all other bacteria. This technique is called streaking for individual colonies. Since bacteria are so small, you will not be able to see that isolated bacterium. However, that bacterium will reproduce itself by binary fission (typical division time is on the order of 20 minutes), resulting in bacteria which are genetically identical to the original bacterium and to each other. These bacteria are visible as a small round colony growing where there had been one isolated bacterium. This method allows you to use the individual colony repeatedly and expect similar results.When innoculating agar plate, Is streaking the plate same as isolation technique? Please clarify.?
Yes in a way - through subsequent streaks around the agar plate you are isolating the bacteria - using differential or selective media will further enhance these colonies!When innoculating agar plate, Is streaking the plate same as isolation technique? Please clarify.?
well, when you innoculate a certain colony and transfer it into other agar plate that is the time you isolate that certain colony.eye make up
Yes in a way - through subsequent streaks around the agar plate you are isolating the bacteria - using differential or selective media will further enhance these colonies!When innoculating agar plate, Is streaking the plate same as isolation technique? Please clarify.?
well, when you innoculate a certain colony and transfer it into other agar plate that is the time you isolate that certain colony.
If you have been removed from people with different experiences reflect circumstances that made this isolation?
John Mark Karr (born December 11, 1969) was a leading pediatric psychiatrist (and occasional entertainer) at the Mayonnaise Clinic in Bangkok, Thailand. He was most noted for developeding several breakthrough techniques in child reering, such as his patented ';hug of love around the neck with a rope';.
He was recently found not guilty for the 1996 robbery of beauty pageant queen JonBen茅t Ramsey. It is known that Karr struggled with urges of kleptomania his entire life, the rest of which will be spent in prison.
Karr was raised in the suburbs of Boulder, Colorado. He was the 12th of 7 children, and led a normal life. After graduating at the bottom of his class in high school, he moved out of Boulder and went to Thailand to study in education.
While studying at the University of Goa Tse in Bangkok, Karr met his soon-to-be wife, 5 yr. old Quientana Shotts [1]. The couple married only weeks after meeting, but lasted less than three years. When Shotts turned 8, Karr divorced her and hastily married 4 yr. old Lara Knutson. Knutson and Karr are still happily married to this day, and Knutson is still 4 years old.
John Mark Karr stayed in Thailand after acquiring his much sought-after degree, and began teaching at several elementary schools in and around the Bangkok area.
Karr taught for several years until he was accused of stealing from several students, in which he was relieved of his teaching duties. Karr later confessed openly to the public that he stole from his students, and was prosecuted several times in the early 90's after police found pirated music on his computer. He was charged in 1992 for the crime, and was sentenced to two years of probation and voluntarily submitted himself to a psychiatric ward for his life-long struggle with kleptomania.
While in the ward, Karr undertook independent studies to become a pediatric psychologist, which many suspect was an influence from his time at the ward.
After only four years of rehabilitation, Karr was invited to a Christmas party by his long-time neighbors, the Ramseys. The occasion was not just to celebrate Christmas, but to celebrate Karr's release from the ward and his earning of a degree in child psychology.
He was recently found not guilty for the 1996 robbery of beauty pageant queen JonBen茅t Ramsey. It is known that Karr struggled with urges of kleptomania his entire life, the rest of which will be spent in prison.
Karr was raised in the suburbs of Boulder, Colorado. He was the 12th of 7 children, and led a normal life. After graduating at the bottom of his class in high school, he moved out of Boulder and went to Thailand to study in education.
While studying at the University of Goa Tse in Bangkok, Karr met his soon-to-be wife, 5 yr. old Quientana Shotts [1]. The couple married only weeks after meeting, but lasted less than three years. When Shotts turned 8, Karr divorced her and hastily married 4 yr. old Lara Knutson. Knutson and Karr are still happily married to this day, and Knutson is still 4 years old.
John Mark Karr stayed in Thailand after acquiring his much sought-after degree, and began teaching at several elementary schools in and around the Bangkok area.
Karr taught for several years until he was accused of stealing from several students, in which he was relieved of his teaching duties. Karr later confessed openly to the public that he stole from his students, and was prosecuted several times in the early 90's after police found pirated music on his computer. He was charged in 1992 for the crime, and was sentenced to two years of probation and voluntarily submitted himself to a psychiatric ward for his life-long struggle with kleptomania.
While in the ward, Karr undertook independent studies to become a pediatric psychologist, which many suspect was an influence from his time at the ward.
After only four years of rehabilitation, Karr was invited to a Christmas party by his long-time neighbors, the Ramseys. The occasion was not just to celebrate Christmas, but to celebrate Karr's release from the ward and his earning of a degree in child psychology.
Any one know any paintings which express isolation?
Check out http://www.piotrwolodkowicz.com and the painting is called a drunk. It is of an emotionally and lonely person left with nothing but his drink.Any one know any paintings which express isolation?
There is few from Picasso's blue periodAny one know any paintings which express isolation?
Gustave Caillebotte (French, 1848-1894)
On the Pont de l'Europe 1876-77
Man, that's one cold, lonely image. Its like being there with a cheap camera on a cold winter day.
http://www.kimbellart.org/database/index鈥?/a>
One famous one can be The Scream by Edvard Munch. In the painting the person screaming is portrayed as alone. When looking closely at the picture everything else is in pairs. The two people on the bridge and the two boats in the water. The sky gives an eerie feeling of loneliness. I believe this is best suited for isolation.
a lot of Edward Hoppers work shows this
the scream
There is few from Picasso's blue periodAny one know any paintings which express isolation?
Gustave Caillebotte (French, 1848-1894)
On the Pont de l'Europe 1876-77
Man, that's one cold, lonely image. Its like being there with a cheap camera on a cold winter day.
http://www.kimbellart.org/database/index鈥?/a>
One famous one can be The Scream by Edvard Munch. In the painting the person screaming is portrayed as alone. When looking closely at the picture everything else is in pairs. The two people on the bridge and the two boats in the water. The sky gives an eerie feeling of loneliness. I believe this is best suited for isolation.
a lot of Edward Hoppers work shows this
the scream
I need help with somebody named Isolation 2020?
I feel really bad about this, but I have a friend on Yahoo who is named Isolation 2020 that I talked to once. Now, he is online, but he doesn't reply when I respond to him. Is he ignoring me, or is he just not at the computer, but keeps his YIM on. He said he was in the army also. Can anyone help me? Or if anybody knows him?I need help with somebody named Isolation 2020?
He might be ignoring you or he might not be at his computer. Shoot him an email, if he never responds then he is ignorning you and you should just move on.
He might be ignoring you or he might not be at his computer. Shoot him an email, if he never responds then he is ignorning you and you should just move on.
When you are a carrier of MRSA should you be allowed to be around other people or in isolation? Why?
here at the hospital, mrsa patients are in isolation. Once MRSA, always MRSAWhen you are a carrier of MRSA should you be allowed to be around other people or in isolation? Why?
In hospital acquired MRSA, people routinely are isolated ( on contact isolation) because most people in the hospital have a comprimised immune system and more suseptible to infection. This is especially true for people who are on antibiotics because MRSA is an infection which is highly resistant to antibiotics. People who are healthy are generally not as suseptible, but should practice regular handwashing to prevent the transmission to others who are ill.
They are rountinely placed on contact precautions because this is how the infection is transmitted...by touching infected materials. They may be cohorted with other patients who have MRSA but no other contaiguous infections.
Here are the CDC recommendations:
http://www.cdc.gov/ncidod/dhqp/ar_mrsa_h鈥?/a>When you are a carrier of MRSA should you be allowed to be around other people or in isolation? Why?
when not in a hospital setting-it's not necessary to remain in isolation and yes you can be around people. if you are a carrier, it means the bacteria is colonized and not currently active. good hygeine and handwashing is all that is necessary.
in the hospital setting, anytime a patient is admitted that is a carrier of MRSA-they are checked to see if the bacteria is active or not-if it is active, they are placed in contact isolation which requires anyone entering the room to wear special gowns and gloves to protect them from the MRSA. these patients can only be put into rooms with other active MRSA carriers or by themselves. the reason for this is b/c everyone in the hospital has a compromised immune system and the disease can be contracted easily.
non-active carriers do not pose a threat to anyone they are around. this includes children and pregnant women. just good handwashing and regular checkups with the doc will help control the spread.
MRSA is highly infectious and belong to the contact isolation category. In other words, MRSA is spread by contact and therefore require isolation. In the USA nurses providing care for MRSA patients are required to wear protective gears like gloves and gown so that they do not spread MRSA to other patients and health care providers.
You can be a carrier of MRSA and be unaware of it. People with MRSA should usually be isolated (depending on where the infection is located i.e. urine, sputum, etc) If it is in the sputum and the person has a productive cough then I would wear a mask gloves and an isolation gown and of course the person would be in isolation. In the urine it is ok to share a room if you use your own commode. They are mostly in isolation though, and usually are on many weeks of IV drug therapy.
You should be in isolation because it is highly contagious. Any one around you should use universal precautions ie. gloves, mask, we even put on certain kinds of gowns so that it doesn't get on our clothes.
In hospital acquired MRSA, people routinely are isolated ( on contact isolation) because most people in the hospital have a comprimised immune system and more suseptible to infection. This is especially true for people who are on antibiotics because MRSA is an infection which is highly resistant to antibiotics. People who are healthy are generally not as suseptible, but should practice regular handwashing to prevent the transmission to others who are ill.
They are rountinely placed on contact precautions because this is how the infection is transmitted...by touching infected materials. They may be cohorted with other patients who have MRSA but no other contaiguous infections.
Here are the CDC recommendations:
http://www.cdc.gov/ncidod/dhqp/ar_mrsa_h鈥?/a>When you are a carrier of MRSA should you be allowed to be around other people or in isolation? Why?
when not in a hospital setting-it's not necessary to remain in isolation and yes you can be around people. if you are a carrier, it means the bacteria is colonized and not currently active. good hygeine and handwashing is all that is necessary.
in the hospital setting, anytime a patient is admitted that is a carrier of MRSA-they are checked to see if the bacteria is active or not-if it is active, they are placed in contact isolation which requires anyone entering the room to wear special gowns and gloves to protect them from the MRSA. these patients can only be put into rooms with other active MRSA carriers or by themselves. the reason for this is b/c everyone in the hospital has a compromised immune system and the disease can be contracted easily.
non-active carriers do not pose a threat to anyone they are around. this includes children and pregnant women. just good handwashing and regular checkups with the doc will help control the spread.
MRSA is highly infectious and belong to the contact isolation category. In other words, MRSA is spread by contact and therefore require isolation. In the USA nurses providing care for MRSA patients are required to wear protective gears like gloves and gown so that they do not spread MRSA to other patients and health care providers.
You can be a carrier of MRSA and be unaware of it. People with MRSA should usually be isolated (depending on where the infection is located i.e. urine, sputum, etc) If it is in the sputum and the person has a productive cough then I would wear a mask gloves and an isolation gown and of course the person would be in isolation. In the urine it is ok to share a room if you use your own commode. They are mostly in isolation though, and usually are on many weeks of IV drug therapy.
You should be in isolation because it is highly contagious. Any one around you should use universal precautions ie. gloves, mask, we even put on certain kinds of gowns so that it doesn't get on our clothes.
I hv to do a project on protoplast isolation from plant source,from where do i procure cellulase n viscozyme ?
please help me out !!
from where do i procure these enzymes??(from delhi)
my school lab doesnot provide me with these enzymeI hv to do a project on protoplast isolation from plant source,from where do i procure cellulase n viscozyme ?
Cellulases Onozuka R-10 Trichoderma viride Kinki Yokult Mfg. Co. Ltd. 8-12, Shingikancho, Nishinomiya, Japan
Cellulases Onozuka RS T. viride Yokult Honsha Co., Tokyo, Japan
Cellulases YC T. viride Seishin Pharma Co. Ltd. 9-500-1, Nagareyama Nagareyama-shi Chiba-ken, Japan
Cellulases CEL T. viride Cooper Biomedica Inc. Malvem, PA, USA
Cellulysin T. viride Calbiochem, San Diego, CA,USA
Driselase Irpex lacteus Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan
Meicelase P-1 T. viride Meiji Seiki Kaisha Ltd., No.8, 2-Chome' Kyobashi, Chou-Ku, Japan
Hemicellulases
Helicase Helix pomatia Industrie Biologique Francaise, Gennevilliers, France
Hemicellulases Aspergillus niger Sigma Chemical Co., St. Louis, MO,USA
Hemicellulases H-2125 Rhizopus sp. Sigma Chemical Co., Munieh, Germany
Rhozyme HP 150 Aspergillus niger Genencor Inc., San Francisco, CA, USA
Pectinases
Macerase Rhizopus arrhizus Calbiochem, San Diego, CA,USA
Macerozyme R -10 R. arrhizus Yakult Hosha Co., Tokyo, Japan
PATE Bacillus polymyxa Farbwerke-Hoechst AG Frankfurt, Germany
Pectinol Aspergillus sp. Rohm and Haas Co. Independence Hall West, Philadelphia, PA 19105, USA
Pectolyase Y-23 Aspergillus japonicus Seishin Pharma Co.,Ltd., Tokyo, Japan
Zymolyase Arthrobacter luteus Sigma Chemical Co., Ltd., St. Louis, MO, USA
Address : 454, FIE, PATPARGANJ INDUSTRIAL AREA, DELHI - 110092, INDIA
Phone : 91-11-32498616/32498099/32666267
Mobile : +919334793446
Fax : 91-11-22161484
Address : 6/14, KIRTI NAGAR INDUSTRIAL AREA, NEW DELHI - 110015, INDIA
Phone : 91-11-45854585
Mobile : +919958155133
Fax : 91-11-45854500eye make up
from where do i procure these enzymes??(from delhi)
my school lab doesnot provide me with these enzymeI hv to do a project on protoplast isolation from plant source,from where do i procure cellulase n viscozyme ?
Cellulases Onozuka R-10 Trichoderma viride Kinki Yokult Mfg. Co. Ltd. 8-12, Shingikancho, Nishinomiya, Japan
Cellulases Onozuka RS T. viride Yokult Honsha Co., Tokyo, Japan
Cellulases YC T. viride Seishin Pharma Co. Ltd. 9-500-1, Nagareyama Nagareyama-shi Chiba-ken, Japan
Cellulases CEL T. viride Cooper Biomedica Inc. Malvem, PA, USA
Cellulysin T. viride Calbiochem, San Diego, CA,USA
Driselase Irpex lacteus Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan
Meicelase P-1 T. viride Meiji Seiki Kaisha Ltd., No.8, 2-Chome' Kyobashi, Chou-Ku, Japan
Hemicellulases
Helicase Helix pomatia Industrie Biologique Francaise, Gennevilliers, France
Hemicellulases Aspergillus niger Sigma Chemical Co., St. Louis, MO,USA
Hemicellulases H-2125 Rhizopus sp. Sigma Chemical Co., Munieh, Germany
Rhozyme HP 150 Aspergillus niger Genencor Inc., San Francisco, CA, USA
Pectinases
Macerase Rhizopus arrhizus Calbiochem, San Diego, CA,USA
Macerozyme R -10 R. arrhizus Yakult Hosha Co., Tokyo, Japan
PATE Bacillus polymyxa Farbwerke-Hoechst AG Frankfurt, Germany
Pectinol Aspergillus sp. Rohm and Haas Co. Independence Hall West, Philadelphia, PA 19105, USA
Pectolyase Y-23 Aspergillus japonicus Seishin Pharma Co.,Ltd., Tokyo, Japan
Zymolyase Arthrobacter luteus Sigma Chemical Co., Ltd., St. Louis, MO, USA
Address : 454, FIE, PATPARGANJ INDUSTRIAL AREA, DELHI - 110092, INDIA
Phone : 91-11-32498616/32498099/32666267
Mobile : +919334793446
Fax : 91-11-22161484
Address : 6/14, KIRTI NAGAR INDUSTRIAL AREA, NEW DELHI - 110015, INDIA
Phone : 91-11-45854585
Mobile : +919958155133
Fax : 91-11-45854500
What was Erickson's stage of intimacy versus isolation?
Young adulthood.What was Erickson's stage of intimacy versus isolation?
It is stage six.
It is stage six.
Define and distinguish between system and isolation system?
It would help if you put this in context.
I would guess that the isolation system is self contained and separated from outside factors.
I would guess that the isolation system is self contained and separated from outside factors.
Where can i find the names of all known components of plant Isolation?regarding organic chemistry...?
so that i ll search for the 5 unknown components for my research...n nt research for the already known ones...my research is on IsolationWhere can i find the names of all known components of plant Isolation?regarding organic chemistry...?
Holy smokes, that is a broad topic! - I would narrow it down a little bit if I were you to a specific class of compounds or drugs, otherwise, you are going to get bogged down is a mess of literature that is going to make your head spin around like the girl from ';The Exorcist';.Where can i find the names of all known components of plant Isolation?regarding organic chemistry...?
good answer but not much help...thanks anyways Report Abuse
You can look it up using the Chemical Abstracts or better yet, if you have access to it, is SciFinder (university libraries should have both of these). From either one of these you will easily be able to find information on any compounds if interest.
Holy smokes, that is a broad topic! - I would narrow it down a little bit if I were you to a specific class of compounds or drugs, otherwise, you are going to get bogged down is a mess of literature that is going to make your head spin around like the girl from ';The Exorcist';.Where can i find the names of all known components of plant Isolation?regarding organic chemistry...?
good answer but not much help...thanks anyways Report Abuse
You can look it up using the Chemical Abstracts or better yet, if you have access to it, is SciFinder (university libraries should have both of these). From either one of these you will easily be able to find information on any compounds if interest.
What are the major steps in isolation of subcellular components?
the steps in this isolation mainly are
1. Centrifugation of cell culture
2. Homogenization at 4 degree
3. PNS
4. high speed sedimentation which
5. collect soluble proteins and membranes
6.sucrose gradient floatation which
7. collect intact organelles and vesicles
8.collection of steps 5 and 7 are tested under electron microscopes for quality control
9. protein fractation with sodium carbonate
10. organelles analysis ELECTROPHORESIS AND CHROMATOGRAPHYWhat are the major steps in isolation of subcellular components?
Firstly, diagram the correlation of the components in question. Then prioritize the substrains of each components, ensuring that they are extractable in their natural cellular form. When this is done, and I must emphasize that it be done first, then and only then can you actually extract the desired component from the rest of the field.
Good luck!What are the major steps in isolation of subcellular components?
Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules, is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.
In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have shown their usefulness.
The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid, milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s) can be eluted and used for further work.
Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In some cases (e.g., isolation of intracellular proteins) it is even possible to integrate the disintegration and separation steps and thus shorten the total separation time [1]. Due to the magnetic properties of magnetic adsorbents (and diamagnetic properties of majority of the contaminating molecules and particles present in the treated sample), they can be relatively easily and selectively removed from the sample. In fact, magnetic separation is the only feasible method for recovery of small magnetic particles (diameter ca 0.1 鈥?1 渭m) in the presence of biological debris and other fouling material of similar size. Moreover, the power and efficiency of magnetic separation procedures is especially useful at large-scale operations. The magnetic separation techniques are also the basis of various automated procedures, especially magnetic-particle based immunoassay systems for the determination of a variety of analytes, among them proteins and peptides. Several automated systems for the separation of proteins or nucleic acids have become available recently.
Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation procedure [2]. Both the reduced shearing forces and the higher protein concentration throughout the isolation process positively influence the separation process.
Separation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation etc. [3].
The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select the optimal magnetic material and the purification procedure.
Necessary materials and equipment
The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations, but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.
Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used. Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10 渭m. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully used [4]. Magnetic particles having the diameter larger than ca 1 渭m can be easily separated using simple magnetic separators, while separation of smaller particles (magnetic colloids with the particle size ranging between tens and hundreds of nanometers) may require the usage of high gradient magnetic separators.
Commercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere [5-7].
Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies, protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised (e.g., antibodies in the case of immobilised protein A, protein G or secondary antibodies, biotinylated molecules in the case of immobilised streptavidin).
Also some other affinity ligands (e.g., nitrilotriacetic acid, glutathione, trypsin, trypsin inhibitor, gelatine etc.) are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NH2 are used for immobilisation, in some cases magnetic particles are available already in the activated form (e.g., tosylactivated, epoxyactivated etc).
In the laboratory magnetite (or similar magnetic materials such as maghemite or ferrites) particles can be surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become available, which enable easy immobilisation of affinity ligands [8]. In exceptional cases enzyme activity can be decreased as a result of usage of magnetic particles with exposed iron oxides. In this case encapsulated microspheres, having an outer layer of pure polymer, will be safer.
Biopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is mechanically broken into fine particles [9]. Alternatively biopolymer solution containing dispersed magnetite is dropped into a mixed hardening solution [4] or water-in-oil suspension technique is used to prepare spherical particles [10].
Basically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide, poly(vinylalcohol) and many others [11].
In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus modifying this material into magnetic form [12,13]. Alternatively magnetic Sepharose or other agarose gels were prepared by simple contact with freshly precipitated or finely powdered magnetite [12,14].
Magnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of specific proteins, have the potential for the separation of antiphospholipid antibodies [15], IgG antibodies [16] and other proteins of interest [17].
Recently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material for specific separation of target proteins [18].
Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes, some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to approx. 500 鈥?1000 ml). Examples of typical batch magnetic separators are shown in Fig. 1.
Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.
For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for the separation of magnetic affinity adsorbents from litre volumes of suspensions was described [19].
Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand [20]. The commercially available platforms can be obtained from several companies such as Qiagen, USA (BioRobot and BioSprint series), Tecan, Japan (Te-MagS) or Thermo Electron Corporation, USA (KingFisher).
Basic principles of magnetic separations of proteins and peptides
Magnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful to obtain good results.
Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.
Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation, magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.
In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies, protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods, magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific applications (especially in molecular biology, bioanalytical chemistry or environmental chemistry) they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.
The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.
In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with diameters above ca 1 渭m) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.
Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used. The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be reached [21,22].
Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize the bed is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations [23].
Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme activity [24,25].
Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials. In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of ionic strength, use of polarity reducing agents (e.g., dioxane or ethyleneglycol) or the use of deforming eluents containing chaotropic salts. Affinity elution (e.g., elution of glycoproteins from lectin coated magnetic beads by the addition of free sugar) may be both a very efficient and gentle procedure.
Examples of magnetic separations of proteins and peptides
Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc [26-29]. Tables 1, 2, 3, 4, 5, 6, 7, 8, 9 show some selected applications of these techniques for proteins and peptides isolation.
In the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells [5] and can thus be used for the separation of specific proteins.
Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides can also be used for further mass spectroscopy characterization [30,31]. The basic principles of magnetic separations can be used in the course of protein or peptide determination using various types of solid phase immunoassays. Usually immunomagnetic particles directly capture the target analyte, or magnetic particles with immobilised streptavidin are used to capture the complex of biotinylated primary antibody and the analyte. The separated analyte is then determined (usually without elution) using an appropriate method. A combination of magnetic separation with affinity capillary electrophoresis is also possible [32].
Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers can be used (see Tables 1, 2, 3, 4).
Genetic engineering enables the construction of gene fusions resulting in fusion proteins having the combined properties of the original gene products. To date, a large number of different gene fusion systems, involving fusion partners that range in size from one amino acid to whole proteins, capable of selective interaction with a ligand immobilized onto magnetic particles or chromatography matrices, have been described. In such systems, different types of interactions, such as enzyme-substrate, receptor-target protein, polyhistidines-metal ion, and antibody-antigen, have been utilized. The conditions for purification differ from system to system and the environment tolerated by the target protein is an important factor for deciding which affinity fusion partner to choose. In addition, other factors, including protein localization, costs for the affinity matrix and buffers, and the possibilities of removing the fusion partner by site-specific cleavage, should also be considered [33,34]. As an example, isolation of recombinant oligohistidine-tagged proteins is based on the application of metal chelate magnetic adsorbents [35,36]. This method has been used successfully for the purification of proteins expressed in bacterial, mammalian, and insect systems.
Antibodies from ascites, serum and tissue culture supernatants can be efficiently isolated using magnetic particles with immobilized Protein A, Protein G or anti-immunoglobulin antibodies. Protein A, isolated from Staphylococcus aureus, binds the Fc region of IgG of most mammalian species with high affinity, leaving antigen specific sites free. Protein G, isolated from Streptococcus sp., reacts with a larger number of IgG isotypes. It has a higher binding affinity to immunoglobulins than Protein A, however, it also interacts with the Fab regions of IgG, although the affinity is ten times lower than for the Fc region [37]. Antiphospholipid antibodies were successfully isolated using magnetoliposomes [15].
Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers binding proteins can be immobilised to magnetic particles and used for isolation of target proteins.
DNA/RNA binding proteins (e.g., promoters, gene regulatory proteins and transcription factors) are often short-lived and in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually eluted with high salt buffer or change of pH [38].
Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPSc from its conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [39,40].
Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies, involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields [41].
A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been described recently [31]. Magnetite particles were covered with a gold layer and then stabilized with ionic agents. These charged stabilizers present at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins and peptides from aqueous solutions. The species trapped by the isolated particles were then characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) after a simple washing.
Magnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular weight xenobiotics [42,43] but using suitable magnetic adsorbents the MSPE could be used to preconcetrate target proteins and peptides as well.
Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from Xenopus egg extracts for analyses of the cell mitosis mechanisms [44,45]. Rapid removal of contaminating proteolytic enzymes from the crude samples could increase yields of sensitive proteins due to the limitation of their proteolysis [46].
A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity [1].
An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic vesicles of Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated and then disrupted to release proteins which were resolved by SDS-PAGE. After 鈥瀒n-gel鈥?digestion with endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles [47]. Analogous procedure was used for the separation and study of peroxisomes proteins when at first peroxisomes were separated using magnetic beads with immobilized specific antibodies and then the protein content of the separated peroxisomes was analysed [48].
Conclusions
Standard liquid column chromatography is currently the most often used technique for the isolation and purification of target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity particles are currently used mainly in molecular biology (especially for nucleic acids separation), cell biology and microbiology (separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA and related techniques (especially determination of clinical markers and environmental contaminants). Up to now separations in small scale prevail and thus the full potential of these techniques has not been fully exploited.
It can be expected that further development will be focused at least on two areas. The first one will be focused on the laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies, such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes capable of magnetic separation and detection of extremely small amount of target biologically active compounds [49].
In the second area, larger-scale (industrial) systems are believed to be developed and used for the isolation of biologically active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps (clarification, concentration and initial purification) into a single unit operation [50]. It is not expected that extremely large amounts of low cost products will be isolated using magnetic techniques, but the attention should be focused onto the isolation of minor, but highly valuable components present in raw materials. Of course, prices of magnetic carriers have to be lowered and special types of low-cost, biotechnology applicable magnetic carriers and adsorbents prepared by simple and cheap procedures have to become available. The existence of inexpensive and effective magnetic separators enabling large-scale operations is necessary, as well.
In the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another way for biological materials separation [51].
It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology industry in the near future.
Acknowledgements
The research is a part of ILE Research Intention No. AV0Z6087904. The work was supported by the Ministry of Education of the Czech Republic (Project No. ME 583) and Grant Agency of the Czech Academy of Sciences (Project No. IBS6087204).
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1. Centrifugation of cell culture
2. Homogenization at 4 degree
3. PNS
4. high speed sedimentation which
5. collect soluble proteins and membranes
6.sucrose gradient floatation which
7. collect intact organelles and vesicles
8.collection of steps 5 and 7 are tested under electron microscopes for quality control
9. protein fractation with sodium carbonate
10. organelles analysis ELECTROPHORESIS AND CHROMATOGRAPHYWhat are the major steps in isolation of subcellular components?
Firstly, diagram the correlation of the components in question. Then prioritize the substrains of each components, ensuring that they are extractable in their natural cellular form. When this is done, and I must emphasize that it be done first, then and only then can you actually extract the desired component from the rest of the field.
Good luck!What are the major steps in isolation of subcellular components?
Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules, is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.
In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have shown their usefulness.
The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid, milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s) can be eluted and used for further work.
Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In some cases (e.g., isolation of intracellular proteins) it is even possible to integrate the disintegration and separation steps and thus shorten the total separation time [1]. Due to the magnetic properties of magnetic adsorbents (and diamagnetic properties of majority of the contaminating molecules and particles present in the treated sample), they can be relatively easily and selectively removed from the sample. In fact, magnetic separation is the only feasible method for recovery of small magnetic particles (diameter ca 0.1 鈥?1 渭m) in the presence of biological debris and other fouling material of similar size. Moreover, the power and efficiency of magnetic separation procedures is especially useful at large-scale operations. The magnetic separation techniques are also the basis of various automated procedures, especially magnetic-particle based immunoassay systems for the determination of a variety of analytes, among them proteins and peptides. Several automated systems for the separation of proteins or nucleic acids have become available recently.
Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation procedure [2]. Both the reduced shearing forces and the higher protein concentration throughout the isolation process positively influence the separation process.
Separation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation etc. [3].
The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select the optimal magnetic material and the purification procedure.
Necessary materials and equipment
The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations, but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.
Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used. Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10 渭m. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully used [4]. Magnetic particles having the diameter larger than ca 1 渭m can be easily separated using simple magnetic separators, while separation of smaller particles (magnetic colloids with the particle size ranging between tens and hundreds of nanometers) may require the usage of high gradient magnetic separators.
Commercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere [5-7].
Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies, protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised (e.g., antibodies in the case of immobilised protein A, protein G or secondary antibodies, biotinylated molecules in the case of immobilised streptavidin).
Also some other affinity ligands (e.g., nitrilotriacetic acid, glutathione, trypsin, trypsin inhibitor, gelatine etc.) are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NH2 are used for immobilisation, in some cases magnetic particles are available already in the activated form (e.g., tosylactivated, epoxyactivated etc).
In the laboratory magnetite (or similar magnetic materials such as maghemite or ferrites) particles can be surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become available, which enable easy immobilisation of affinity ligands [8]. In exceptional cases enzyme activity can be decreased as a result of usage of magnetic particles with exposed iron oxides. In this case encapsulated microspheres, having an outer layer of pure polymer, will be safer.
Biopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is mechanically broken into fine particles [9]. Alternatively biopolymer solution containing dispersed magnetite is dropped into a mixed hardening solution [4] or water-in-oil suspension technique is used to prepare spherical particles [10].
Basically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide, poly(vinylalcohol) and many others [11].
In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus modifying this material into magnetic form [12,13]. Alternatively magnetic Sepharose or other agarose gels were prepared by simple contact with freshly precipitated or finely powdered magnetite [12,14].
Magnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of specific proteins, have the potential for the separation of antiphospholipid antibodies [15], IgG antibodies [16] and other proteins of interest [17].
Recently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material for specific separation of target proteins [18].
Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes, some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to approx. 500 鈥?1000 ml). Examples of typical batch magnetic separators are shown in Fig. 1.
Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.
For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for the separation of magnetic affinity adsorbents from litre volumes of suspensions was described [19].
Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand [20]. The commercially available platforms can be obtained from several companies such as Qiagen, USA (BioRobot and BioSprint series), Tecan, Japan (Te-MagS) or Thermo Electron Corporation, USA (KingFisher).
Basic principles of magnetic separations of proteins and peptides
Magnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful to obtain good results.
Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.
Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation, magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.
In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies, protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods, magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific applications (especially in molecular biology, bioanalytical chemistry or environmental chemistry) they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.
The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.
In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with diameters above ca 1 渭m) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.
Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used. The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be reached [21,22].
Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize the bed is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations [23].
Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme activity [24,25].
Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials. In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of ionic strength, use of polarity reducing agents (e.g., dioxane or ethyleneglycol) or the use of deforming eluents containing chaotropic salts. Affinity elution (e.g., elution of glycoproteins from lectin coated magnetic beads by the addition of free sugar) may be both a very efficient and gentle procedure.
Examples of magnetic separations of proteins and peptides
Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc [26-29]. Tables 1, 2, 3, 4, 5, 6, 7, 8, 9 show some selected applications of these techniques for proteins and peptides isolation.
In the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells [5] and can thus be used for the separation of specific proteins.
Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides can also be used for further mass spectroscopy characterization [30,31]. The basic principles of magnetic separations can be used in the course of protein or peptide determination using various types of solid phase immunoassays. Usually immunomagnetic particles directly capture the target analyte, or magnetic particles with immobilised streptavidin are used to capture the complex of biotinylated primary antibody and the analyte. The separated analyte is then determined (usually without elution) using an appropriate method. A combination of magnetic separation with affinity capillary electrophoresis is also possible [32].
Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers can be used (see Tables 1, 2, 3, 4).
Genetic engineering enables the construction of gene fusions resulting in fusion proteins having the combined properties of the original gene products. To date, a large number of different gene fusion systems, involving fusion partners that range in size from one amino acid to whole proteins, capable of selective interaction with a ligand immobilized onto magnetic particles or chromatography matrices, have been described. In such systems, different types of interactions, such as enzyme-substrate, receptor-target protein, polyhistidines-metal ion, and antibody-antigen, have been utilized. The conditions for purification differ from system to system and the environment tolerated by the target protein is an important factor for deciding which affinity fusion partner to choose. In addition, other factors, including protein localization, costs for the affinity matrix and buffers, and the possibilities of removing the fusion partner by site-specific cleavage, should also be considered [33,34]. As an example, isolation of recombinant oligohistidine-tagged proteins is based on the application of metal chelate magnetic adsorbents [35,36]. This method has been used successfully for the purification of proteins expressed in bacterial, mammalian, and insect systems.
Antibodies from ascites, serum and tissue culture supernatants can be efficiently isolated using magnetic particles with immobilized Protein A, Protein G or anti-immunoglobulin antibodies. Protein A, isolated from Staphylococcus aureus, binds the Fc region of IgG of most mammalian species with high affinity, leaving antigen specific sites free. Protein G, isolated from Streptococcus sp., reacts with a larger number of IgG isotypes. It has a higher binding affinity to immunoglobulins than Protein A, however, it also interacts with the Fab regions of IgG, although the affinity is ten times lower than for the Fc region [37]. Antiphospholipid antibodies were successfully isolated using magnetoliposomes [15].
Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers binding proteins can be immobilised to magnetic particles and used for isolation of target proteins.
DNA/RNA binding proteins (e.g., promoters, gene regulatory proteins and transcription factors) are often short-lived and in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually eluted with high salt buffer or change of pH [38].
Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPSc from its conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [39,40].
Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies, involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields [41].
A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been described recently [31]. Magnetite particles were covered with a gold layer and then stabilized with ionic agents. These charged stabilizers present at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins and peptides from aqueous solutions. The species trapped by the isolated particles were then characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) after a simple washing.
Magnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular weight xenobiotics [42,43] but using suitable magnetic adsorbents the MSPE could be used to preconcetrate target proteins and peptides as well.
Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from Xenopus egg extracts for analyses of the cell mitosis mechanisms [44,45]. Rapid removal of contaminating proteolytic enzymes from the crude samples could increase yields of sensitive proteins due to the limitation of their proteolysis [46].
A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity [1].
An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic vesicles of Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated and then disrupted to release proteins which were resolved by SDS-PAGE. After 鈥瀒n-gel鈥?digestion with endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles [47]. Analogous procedure was used for the separation and study of peroxisomes proteins when at first peroxisomes were separated using magnetic beads with immobilized specific antibodies and then the protein content of the separated peroxisomes was analysed [48].
Conclusions
Standard liquid column chromatography is currently the most often used technique for the isolation and purification of target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity particles are currently used mainly in molecular biology (especially for nucleic acids separation), cell biology and microbiology (separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA and related techniques (especially determination of clinical markers and environmental contaminants). Up to now separations in small scale prevail and thus the full potential of these techniques has not been fully exploited.
It can be expected that further development will be focused at least on two areas. The first one will be focused on the laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies, such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes capable of magnetic separation and detection of extremely small amount of target biologically active compounds [49].
In the second area, larger-scale (industrial) systems are believed to be developed and used for the isolation of biologically active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps (clarification, concentration and initial purification) into a single unit operation [50]. It is not expected that extremely large amounts of low cost products will be isolated using magnetic techniques, but the attention should be focused onto the isolation of minor, but highly valuable components present in raw materials. Of course, prices of magnetic carriers have to be lowered and special types of low-cost, biotechnology applicable magnetic carriers and adsorbents prepared by simple and cheap procedures have to become available. The existence of inexpensive and effective magnetic separators enabling large-scale operations is necessary, as well.
In the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another way for biological materials separation [51].
It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology industry in the near future.
Acknowledgements
The research is a part of ILE Research Intention No. AV0Z6087904. The work was supported by the Ministry of Education of the Czech Republic (Project No. ME 583) and Grant Agency of the Czech Academy of Sciences (Project No. IBS6087204).
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