Phospho-specific antibodies as a tool to study in vivo regulation of BRCA1 after DNA damage

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

Characterisation of epitopes of pan-IgG/anti-G3m(u) and anti-Fc monoclonal antibodies.

The characterisation of monoclonal antibodies (MAbs) and their epitopes is important prior to their application as molecular probes. In this study, Western blotting using IgG1 Fc and pFc' fragments was employed to screen seven MAbs before pepscan analysis to determine their reactivity to potentially linear epitopes. MAbs PNF69C, PNF110A, X1A11 and MAbs WC2, G7C, JD312, 1A1 detected epitopes within the C(H)3 and C(H)2 domains, respectively. However, only four MAbs showed pepscan profiles that highlighted likely target residues. In particular, MAbs PNF69C and PNF110A that have previously been characterised with pan-IgG and anti-G3m(u) specificity, detected the peptide motif 338-KAKGQPR-344 which was located within the N-terminal region of the C(H)3 domain. Furthermore the majority of residues were present in all four IgG subclasses. Consequently the peptide identified was consistent with the pan-IgG nature of these antibodies. By using PCImdad, a molecular display programme, this sequence was visualised as surface accessible, located in the C(H)2/C(H)3 inter-domain region and proximal to the residue arginine(435). It is speculated that this residue may be important for phenotypic expression of G3m(u) and specificity of these reagents. Pepscan analysis of MAbs G7C and JD312 (both pan-IgG) highlighted the core peptide sequence 290-KPREE-294, which was present in the C(H)2 domain and was common to all four IgG subclasses. PCImdad also showed this region to be highly accessible and was consistent with previous bioinformatic and autoimmune analysis of IgG. Overall these MAbs may serve as useful anti-IgG or anti-G3m(u) reagents and probes of immunoglobulin structure