Western blots with cell extract from the clear cell renal carcinoma (ccRCC) cell line were incubated with the rabbit polyclonal anti-PARP antibody (Cell Signaling Technology, Cat # 9542) using the traditional manual procedure (A) or BlotCycler™ (B).

The membranes were processed as follows: blocked with TBST-5% dry milk for 1 hour, incubated with primary antibody overnight, washed 3 x 5 minutes with TBST, incubated with the anti-rabbit secondary antibody (Jackson Immunochemicals, 1:4000 dilution) for 1 hour, and washed 3 x 15 minutes with TBST. The bands were visualized using a chemiluminescence substrate.

For the manual procedure, incubation with the primary antibody was carried out at 4°C while the rest of the procedure was performed at room temperature. For the automated procedure, incubation and all processing steps using  BlotCycler™ were done entirely at 4°C.

When the blot was processed using the manual method (A) at room temperature, multiple non-specific bands were observed. In contrast, the blot processed with BlotCycler™ (B) detected only the bands representing the uncleaved PARP protein (113 kD) as well as the C-terminal and N-terminal cleaved PARP fragments (89 kD and 24 kD respectively).

These data suggest the importance of how blocking and washing steps are performed, as well as the temperature maintained during blot washing and antibody incubation. Hands free western blot processing using BlotCycler™ enables researchers to optimize shaking, washing, and antibody incubation to eliminate nonspecific bands and generate high quality results.