Phospho-Specific Antibodies as a Tool to Study In Vivo Regulation of BRCA1 After DNA Damage

摘要: Phospho-specific antibodies have become very useful reagents for study of signal transduction pathways. This chapter describes the production phospho-specific and their use to assess individual phosphorylation events in vivo cells. The first step involves synthesis peptides (12-15 residues), where site is centrally located, a cysteine residue incorporated at either N- or C-terminus peptide facilitate coupling it an immunogenic carrier protein. No special immunization protocols are required generate antibodies. Typically, animals choice immunized twice, several weeks apart, enzyme-linked immunosorbent assay used determine relative titer sera against phosphorylated nonphosphorylated peptides. Where much greater than peptides, can be appropriate dilutions without further processing. In case significant level specific present antisera, enhancement obtain antibody. Although these enhanced antisera suitable many applications, there may circumstances affinity-purified required. These detect particular event using Western blotting, immunoprecipitation, immunofluorescence.