Generation and Characterization of Antibodies Directed Against Di-Modified Histones, and Comments on Antibody and Epitope Recognition

Abstract

This chapter describes the approach and methods used in generating and purifying the polyclonal Phos Ser10/Ac Lys14 di-modified H3 antibody (hereafter Phos/Ac H3 Ab) from rabbit serum. While this particular affinity purification scheme may not be necessary for all antisera, the overall protocol can serve as a general guide for the generation and purification of antibodies against modified histones. The recent advances in the understanding of histone modifications are in part facilitated by the development and widespread uses of site- and modification-specific histone antibodies. Early efforts were focused on generating acetyl histone–specific antibodies to study the links between histone acetylation and a variety of nuclear processes. For example, by immunoblotting analyses, histones acetylated at specific sites are linked to histone deposition, transcription activation, and cell cycle progression. In addition, antibodies to di-modified H3 molecules are also developed to examine the significance of specific combinations of histone modifications. To date, two separate di-modified H3 antibodies have been generated, including one that specifically recognizes H3 phosphorylated at serine 10 (Ser10) and acetylated at lysine 14 (Lys14), the other that specifically recognizes H3 acetylated at Lys9 and phosphorylated at Ser10. In the case of this dimodified H3 antibody, previous studies have shown that H3 phosphorylation in mammalian cells is induced upon epidermal growth factor (EGF) stimulation and correlates with transcription activation of the immediate early genes. In addition to testing the purified antibody by enzyme-linked immunosorbent assay (ELISA), the specificity of histone modification antibodies should also be tested by western blot analyses coupled with peptide competition assays.