The BlotCycler™ operating unattended in 4˚C, produced consistently clearer, more specific bands compared to the manual protocol.
The data suggest the importance of how blocking and washing steps are performed, as well as the temperature of blot washing and antibody incubation. Get a PDF of the application note here.
We compared automated Western blots processed by BlotCycler™ to the traditional manual procedure.
We incubated Western blots with cell extract from the clear cell renal carcinoma (ccRCC) cell line with the rabbit polyclonal anti‐PARP antibody (Cell Signal, 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 3X15 minutes with TBST, incubated with the anti‐rabbit secondary antibody (Jackson Immunochemicals, 1:4000 dilution) for 1 hour, and washed 3X15 minutes 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.
Automated Western Blots processed using BlotCycler™ was done entirely at 4°C. When the blot was processed using the traditional manual method (A), multiple non‐specific bands were observed. In contrast, the blot processed with BlotCycler™ (B) 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) were detected.
These data suggest the importance of how blocking and washing steps are performed, as well as the temperature of blot washing and antibody incubation.
Eliminate nonspecific bands and generate high quality results. Hands-free automated Western Blot using the BlotCycler™ enables researchers and clinicians to optimize shaking, washing, and antibody incubation conditions during western blot processing.