Abstract
Polyacrylamide has been used as reproducible medium than starch for electrophoretic separations. The introduction of a discontinuous buffer system greatly increases sample resolution by electrically forcing proteins in dilute samples to concentrate into zones only microns thick. However, histone proteins readily aggregate in this system. To solve this problem this chapter describes methods for the two-dimensional (2D) gel analysis of histones from yeast and mammals, the protocols are applicable to samples from any organism. Sample preparation, acetic acid and urea (AU) resolving gels and AUC gel preparation, sample electrophoresis, and data analysis are discussed in turn. High-resolution 2D analysis became possible when a discontinuous buffer system was developed for acetic acid, urea, and AU resolving gels. High-resolution 2D analysis showed that several of the unidentified bands on one-dimensional (1D) gels represented variants of the four core nucleosome histone families. In addition, histone acetylated and phosphorylated forms separate from the unmodified forms in this 2D system.
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