Abstract
Publisher Summary This chapter presents several parameters to be taken into consideration and some useful hints for systematic characterization of antibodies raised against methylated histone peptides. It focuses on antibodies against methylated residues—namely, H3-K4, H3-K27, and H4-K20, the methods and procedures described in the chapter are applicable for any antibody directed against the histone tail modifications, including arginine methylation and lysine acetylation, among other modifications. Enzyme-linked immunosorbent assay (ELISA) is the traditional method for antibody characterization. It is the most quantitative and sensitive and the optimal concentration of the antibody. The ELISA should be followed by dot blot analyses. If monoclonal antibodies are to be screened, it is easier to perform dot blot analysis first to isolate specific clones and then proceed to ELISA to ascertain optimal concentrations for the antibody. The antibodies have to be characterized by as many methods as possible and with as many controls as possible. The number of parameters that affect the specificity of the antibody are too many and any result that is derived from chromatin immunoprecipitation (ChIP) and IF experiments must be interpreted with extreme caution.