Anti 53bp1 rabbit polyclonal antibody

Cells were lysed in lysis buffer (Thermo); equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % milk in TBST (20 mM Tris-HCl, 500 mM NaCl [pH7.5], 0.1 % [v/v] Tween 20) for 1 h at room temperature, and incubated with the primary antibodies overnight at 4 °C. Membranes were then incubated with the appropriate secondary antibody for 1 h at room temperature, and washed with TBST. Bands were visualized using the Image quant LAS500 system (GE).
Antibodies used in this study: anti-IER5 goat polyclonal antibody (Abcam), anti-IER5 rabbit polyclonal antibody (Santa Cruz), anti-GAPDH mouse monoclonal antibody (Zhong Shan Jin Qiao), anti-PARP1 rabbit monoclonal antibody (Santa Cruz), anti-Ku70 mouse monoclonal antibody (Abcam), anti-Ku80 rabbit monoclonal antibody (Santa Cruz), anti-FLAG M2 mouse monoclonal antibody (Sigma), anti-53BP1 rabbit polyclonal antibody (Santa Cruz), anti-RAD51 rabbit polyclonal antibody (Proteintech), anti-γH2AX mouse monoclonal antibody (Millipore), anti-Ubiquitinylated proteins mouse monoclonal antibody (Millipore), and anti-PADPR mouse monoclonal antibody (Abcam).

For indirect immunofluorescence staining analysis, 0.3 × 10⁶ transfected cells were plated in 35 mm dishes. Twelve hours after transfection cells were washed with PBS and fixed in 2% paraformaldehyde (PFA) for 15 min. Cells were washed and permeabilized in P-solution (100 mM Tris, pH 7.4, 50 mM EDTA, 0.5% Triton X-100) for 5 min and subsequently incubated in PBG blocking buffer (0.2% gelatin, 0.5% BSA in PBS) at 4°C overnight. The primary antibody was diluted 1:400 in PBG buffer and the cover slips were incubated for 2 h at RT. After washing 3 × 10 min with PBS, cells were incubated for 1.5 h with the secondary antibody diluted 1:400. After DNA counterstaining with 0.025 μg/ml DAPI solution, cells were mounted in 10 μl Promo-fluor antifade mounting media (PromoKine) and processed for scanning on a Confocal Laser Scanning Microscope (TCS SP5, Leica Microsystems). The following antibodies were used: anti-γ-H2AX [3F2] mouse monoclonal antibody (Abcam PLC), anti-53BP1 rabbit polyclonal antibody (Santa Cruz), anti-mouse IgG antibody conjugated with AlexaFluor 488 and anti-rabbit IgG antibody, conjugated with AlexaFluor568 (Life Technologies).