Characterization of BRCA2: temperature sensitivity of detection and cell-cycle regulated expression.
People carrying a mutant BRCA2 gene are susceptible to breast, ovarian, pancreatic and other tumors. Many facets of BRCA2 have been studied, including its mutation in human cancers, its role in mouse embryogenesis and its RNA expression in different tissues and different mouse embryogenesis stages. However, there has been very little characterization of BRCA2 protein. We investigated the biochemical and biological properties of BRCA2 by using a monoclonal antibody we generated against the N-terminal portion of BRCA2. We discovered that the detection of BRCA2 by immunoblot analysis was sensitive to the temperature used to denature the samples before gel electrophoresis. BRCA2 was easily detectable when samples were denatured at low temperature instead of boiling. Although the precise mechanism underlying this observation is not clear yet, this finding will significantly improve our ability to study BRCA2. We examined the expression of BRCA2 using an immunoblot analysis protocol modified according to this observation. We showed that BRCA2 was presented in every human cell lines examined, including Capan-1, which expressed a truncated BRCA2 due to a BRCA2 frameshift mutation. We also showed that the expression of BRCA2 was cell-cycle regulated. Our results suggest that BRCA2 has an important role in cell growth regulation.
Su
L K
LK
Department of Tumor Biology, The University of Texas MD Anderson Cancer Center, Houston 77030, USA.
Wang
S C
SC
Qi
Y
Y
Luo
W
W
Hung
M C
MC
Lin
S H
SH
D000911:Antibodies, Monoclonal; D024682:BRCA2 Protein; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D005260:Female; D006801:Humans; D015151:Immunoblotting; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D024682:BRCA2 Protein; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014157:Transcription Factors