Everything about Electrofocusing totally explained
Isoelectric focusing (IEF), also known as
electrofocusing, is a technique for separating different
molecules by their
electric charge differences. It is a type of zone
electrophoresis, usually performed in a
gel, that takes advantage of the fact that a molecule's charge changes with the
pH of its surroundings. A protein which is in a pH region below its pI will be positively charged and so will migrate towards the cathode. However, as it migrates, the charge will decrease until the protein reaches a pH which is its pI. At this point it has no net charge and so migration ceases. Should it overshoot this point, it'll enter a region of pH above its pI and so become negatively charged. It will then reverse its direction of migration and now migrate towards the anode. Therefore proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.
Molecules to be focused are distributed over a medium that has a pH gradient (usually created by
aliphatic ampholytes). An
electric current is passed through the medium, creating a "positive"
anode and "negative"
cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules
isoelectric point is reached. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such won't proceed any further within the gel. The gradient is initially established before adding the particles of interest by first subjecting a solution of small molecules such as
polyampholytes with varying
pI values to electrophoresis.
The method is applied particularly often in the study of
proteins, which separate based on their relative content of
acidic and
basic residues, whose value is represented by the pI. Proteins are introduced into a
Immobilized pH gradient gel composed of
polyacrylamide,
starch, or
agarose where a pH gradient has been established. Gels with large pores are usually used in this process to eliminate any "sieving" effects, or artifacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can resolve proteins that differ in
pI value by as little as 0.01. Isoelectric focusing is the first step in
two-dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by
molecular weight through
SDS-PAGE.
Further Information
Get more info on 'Electrofocusing'.
|
External Link Exchanges
Do you know how hard it is to get a link from a large encyclopaedia? Well we're different and will prove it. To get a link from us just add the following HTML to your site on a relevant page:
<a href="http://isoelectric_focusing.totallyexplained.com">Isoelectric focusing Totally Explained</a>
Then simply click through this link from your web page. Our crawlers will verify your link, extract the title of your web page and instantly add a link back to it. If you like you can remove the words Totally Explained and embed the link in article text.
As long as your link remains in place, we'll keep our link to you right here. Please play fair - our crawlers are watching. Your site must be closely related to this one's topic. Any kind of spamming, dubious practises or removing the link will result in your link from us being dropped and, potentially, your whole site being banned. |
