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

Khanna, Kum Kum, Gatei, Magtouf and Tribbick, Gordon (2006) Phospho-specific antibodies as a tool to study in vivo regulation of BRCA1 after DNA damage. Methods in Molecular Medicine, 120 441-452. doi:10.1385/1-59259-969-9:441


Author Khanna, Kum Kum
Gatei, Magtouf
Tribbick, Gordon
Title Phospho-specific antibodies as a tool to study in vivo regulation of BRCA1 after DNA damage
Journal name Methods in Molecular Medicine   Check publisher's open access policy
ISSN 1940-6037
Publication date 2006
Sub-type Article (original research)
DOI 10.1385/1-59259-969-9:441
Volume 120
Start page 441
End page 452
Total pages 12
Place of publication Totowa, N.J., U.S.
Publisher Humana Press
Language eng
Subject 03 Chemical Sciences
0304 Medicinal and Biomolecular Chemistry
Abstract 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.
Keyword Antibodies
Phosphorylation Sites
Immunization
Peptides
Q-Index Code C1
Institutional Status Non-UQ
Additional Notes Issue title: Breast Cancer Researh Protocols

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Medicine Publications
 
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Created: Thu, 14 Jan 2010, 15:38:54 EST by Ms May Balasaize on behalf of Faculty Of Health Sciences