Rad51 Protein Analysis in Yeast Strains

Western blottin was performed to check Rad51 levels in NRY1, NRY2, and TSY17 strains. Protein samples were loaded on an SDS polyacrylamide gel. A polyvinylidene difluoride (PVDF) membrane was used for the transfer as described earlier (39 (link)). The primary antibodies used were mouse anti-Act1 (Abcam), rabbit anti-Rad51 (Santa Cruz), and mouse anti-Hsp82 (Calbiochem) at 1:5,000 dilutions. For subcellular fractionation, we used anti-Pgk1 antibody (Novus Biologicals) and mouse anti-Nsp1 antibody (Abcam) at 1:3,000 and 1:5,000 dilutions, respectively. For secondary antibodies, horseradish peroxide-conjugated anti-rabbit antibody (Promega) and anti-mouse antibody (Santa Cruz Biotechnology Inc., CA, USA) were used at 1:10,000 dilutions. The Western blots were developed using a chemiluminescent detection system (Pierce). Every experiment was repeated at least 3 times, and band intensities were quantified by using Image J software. Mean relative densities were plotted using GraphPad prism.