Rat anti-RAD-51 antibody was generated using a His-tagged fusion protein expressed from plasmid pET28a containing the entire RAD-51S coding sequence; immunizations and bleeds were performed by SDIX. Rat anti -RAD-51 was affinity purified against membrane-bound protein as described in Koelle MR and Horvitz (1996) HR with the following modifications: nitrocellulose membrane was blocked in 5% milk in 1×TBST; and, eluates containing rat anti -RAD-51 were further purified by dialysis with 12-14 kDa dialysis tubing (Spectrum) in 1×TBST for 1 hour and overnight at 4°C. Specificity was demonstrated by showing that rat anti-RAD-51 foci colocalize with rabbit anti-RAD-51 foci [21] by immunofluorescence and that these recombination-dependent foci are eliminated in spo-11(me44) gonads.
rad-51