Separation of histone variants and post-translationally modified isoforms by triton/acetic acid/urea polyacrylamide gel electrophoresis.
Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.
Ryan
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Boston College, Chestnut Hill, Massachusetts, USA.
Annunziato
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D019342:Acetic Acid; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006657:Histones / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008567:Membranes, Artificial; D017830:Octoxynol; D011145:Polyvinyls; D020033:Protein Isoforms / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D014508:Urea; D005465:Fluorocarbon Polymers
D019342:Acetic Acid; D003902:Detergents; D006657:Histones; D008567:Membranes, Artificial; D017830:Octoxynol; D011145:Polyvinyls; D020033:Protein Isoforms; D014508:Urea; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers