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Isoelectric focusing is a method of separating proteins based on their relative content of acidic and basic residues. This content is represented by a value known as an isoelectric point, or pI. Proteins are introduced into a gel (composed of polyacrylamide, starch, agarose, etc.) which has an established pH gradient or is capable of establishing such a gradient after applying an electrical current. This gradient is established by subjecting a mixture of polyampholytes, small polymers that have different pI values, to electrophoresis before the application of proteins. Also, the pores of the gel are made to be very large so as to eliminate the sieving effect. Proteins that have been introduced into the gel begin to move until they reach a place in the gel where the pH is equal to the isoelectric point of the protein. Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01.
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