Anti-acetyl-Histone H4, hyperacetylated

Autoantibodies to histone antigens have been described in patients with idiopathic and drug-induced SLE, rheumatoid arthritis, and other conditions. The presence of autoantibodies to histones are frequently found in several rheumatic disorders (1). In one study, the predominant responses to histones in SLE sera were to H1, H2b, and H3. Marked elevations of binding occurred to H1 and H2b in 33% of patients, while 25% showed higher binding to H3 (2). The same study showed the highest anti-histone reactivity to be in patients with rheumatoid arthritis with vasculitis, while the highest reactivity in SLE sera was in those patients with a history of photosensitivity (3). In diploid eukaryotic cells, the chromatin fibers are about 20nM in diameter. They consist of two major components in equal amounts, DNA and basic proteins called histones. The histones are a group of water and dilute acid soluble basic proteins found associated with DNA in chromosomes. They are characterized by relatively high levels of lysine and arginine. Although histones are classified into a limited number of types of fractions (see below) with each particular fraction having a fundamentally distinct amino acid composition and sequence, numerous subfractions are observed due to the acetylation, methylation, and phosphorylation of various amino acid residues. Microheterogeneity or alteration of structure is dynamic such that the histones of a single cell type are found to vary during development. They are believed to play a role in gene activity and cellular metabolism. , Histones are believed to be regularly arranged in the deep groove of the DNA helix. The recurring positive charges of the histones form electrostatic associations with the negatively charged phosphate groups of DNA making the DNA more stable and flexible. This allows for the supercoiling of the chromatin fibers. With the exception of H1, the primary structures of the calf thymus histones have been determined. Comparisons with the structures for histones from other sources indicate that the histones rank among the most highly conserved (low mutation rate) proteins in nature. , Molecular Weights of Histones:, Lysine Rich (H1, f1): ~ 21,500 , Slightly Lysine Rich (H2a, f2a2): 14,004 , Slightly Lysine Rich (H2b, f2b): 13,774 , Arginine Rich (H3, f3): 15,324 , Arginine Rich (H4, f2a1): 11,282, Applications: Suitable for use in Western Blotting, Immunoprecipitation, Immunocytochemistry. Western Blotting: 0.5-2ug/ml will detect acetylated histone H4 in acid-extracted proteins from HeLa cells treated with 5mM sodium butyrate, a previous lot detected acetylated histone H4 in Tetrahymena macronuclei. Sodium butyrate, an inhibitor of deacetylases, was used to enhance detection of acetylated histone H4. Immunoprecipitation: 5ug will immunoprecipitate acetylated histone H4 from 500ug of acid extracted proteins from HeLa cells treated with 5mM sodium butyrate for 24 hours. Immunocytochemistry: 5-10ug/ml gave positive nuclear immunostaining in human A431 cells treated with sodium butyrate and fixed with ice-cold 1% paraformaldehyde in PBS. , Immunoblot Analysis. Acid-extracted proteins from normal HeLa cells and HeLa cells treated with 5mM sodium butyrate for 24 hours were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-acetylated Histone H4 (1ug/ml). Proteins were visualized using a goat-anti rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.