Acid-Urea-Triton Polyacrylamide Gels for Histones

Abstract

Acid-urea polyacrylamide gels are capable of separating basic histone proteins provided they differ sufficiently in size and/or effective charge (see Chapter 14). Separation between similarly sized and charged H2A, H2B, and H3 forms of most organisms can typically not be achieved. Zweidler discovered that core histones but not linker histones or any other known protein (see Note 1) bind the nonionic detergent Triton (1). This limits the usability of detergent addition to the separation of core histones only, unless they must be separated from other basic proteins in an acid-urea gel electrophoresis environment. The binding of Triton to a core histone increases the effective mass of the protein within the gel without affecting its charge, and thus reduces its mobility during electrophoresis. Separation between most or all core histone proteins of diverse species can virtually always be obtained by adjusting concentrations of Triton and of urea, which appears to act as a counter-acting, dissociating agent (2). Experimentally, an optimal balance can be determined by gradient gel electrophoresis with a gradient of urea (3) or Triton (4). The Triton gradient protocol in the discontinuous gel system, developed by Bonner and coworkers (5), is described in Section 3. It has a distinct advantage over the urea gradient protocol. Generally, it can identify a core histone protein band as belonging to histone H4, H2B, H3, or H2A. In this order, apparent affinities for Triton X-100 increase sharply (4,6,7). An example of such a separation of a crude mixture of histones with nonhistone proteins from a tobacco callus culture is shown in Fig. 1A. In addition, a detailed working protocol for a long acid-urea-Triton (AUT) gel at 9 mM Triton and 8M urea is provided. It describes the protocol used extensively in my laboratory for the analysis of core histones, especially of histone H3, dicots (6), monocots (7), and the green alga Chlamydomonas (8). Figure 1B shows an example of the differentially acetylated histone H3 variant proteins of tobacco, purified by reversed-phase HPLC (6). The protocol description directly parallels the acid-urea gel protocol described in Chapter 14, which also provides details for the use of different gel dimensions.