Abstract
This unit describes isoelectric focusing (IEF) of polypeptides in urea-containing polyacrylamide slab gels as a result of a pH gradient created by ampholytes (small charged organic molecules) in the gel in response to an electric field. Separation on the basis of isoelectric point produces bands of polypeptides that can be electrotransferred to suitable membranes for further analysis, for example, by staining or immunodetection. If samples have been radiolabeled, they can be visualized by fluorography. This unit contains support protocols detailing special conditions for application of denaturing IEF slab gels for electrotransfer and fluorography in which a scintillator is incorporated into the gel after completion of the run to facilitate detection of radioactively labeled polypeptides.
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