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Guide to Isoelectric Focusing

APB, RW 5/5/981 Guide to Isoelectric FocusingIntroductionIsoelectric Focusing methods are widely applied for the separation of proteins, peptides andenzymes. The principle: In a pH gradient the sample components migrate towards the anodeor the cathode to the pH values, where their net charges are zero: their Isoelectric points (pI).Should a protein diffuse away from its pI, it would gain a charge and migrate back: this is thefocusing effect. Much more details of the theory and practice of Isoelectric Focusing can befound in the book by Righetti [1] .The proteins are driven to their Isoelectric points by the electric field. The Isoelectric pointsof the proteins can be estimated with a calibration curve using marker proteins. Native ordenaturing conditions can be chosen by omitting or adding high amounts of proteins become highly concentrated at their pIs.

Separations in agarose gels are more rapid than in polyacrylamide gels. The pore size diameter of a 1 % agarose gel is ca. 150 nm. In addition macromolecules larger than 500 kDa can be separated since agarose pores are substantially larger than those of polyacrylamide gels. One oft the reasons to use agarose gels for isoelectric focusing is ...

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jgi.doe.gov

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