Purification and analysis of variant and modified histones using 2D PAGE

Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) systems employing combinations of acetic acid/urea (AU), acetic acid/urea/Triton X-100 (AUT) and sodium dodecyl sulfate (SDS) gel formulations are uniquely effective for resolution of histone variants and their modified derivatives. Coupled with Western transfer methods using modification-specific antibodies and recent advances in mass spectrometry, 2D PAGE emerges as a versatile tool for histone purification and analysis. This chapter describes 2D PAGE gel systems appropriate for histone proteins, including detailed procedures for designing, running, and staining gels. Methods for electrophoretic transfer of histones from AUTxSDS and AUTxAU 2D gels are described and evaluated. Alternatively, methods are provided for obtaining highly purified protein samples from fixed and stained gels via electroelution of proteins from specific gel spots.