Western blot processed by BlotCycler as compared to the traditional manual procedure. Western blots with cell extract from the clear cell renal carcinoma(ccRCC) cell line were incubated with the rabbitpolyclonal anti‐PARP antibody using the traditional manual procedure (A) or BlotCycler(B). The membranes were processedas follows: blocked with TBST‐5% dry milk for 1hour, incubated with primary antibody overnight,washed 3X15 minutes with TBST, incubated withthe anti‐rabbit secondary antibody for 1 hour,and washed 3X15 minutes TBST. The bands werevisualized using a chemiluminescence substrate.aFor the manual procedure, incubation with theprimary antibody was carried out at 4°C while the rest of the procedure was performed at room temperature. Processing using BlotCycler was done entirely at 4°C. When the blot was processed using thetraditional manual method (A), multiple non‐specificbands were observed. In contrast, the blot processedwith BlotCycler(B) were detected only the bandsrepresenting the uncleaved PARP protein (113 kD) aswell as the C‐terminal and N‐terminal cleaved PARPfragments (89 kD and 24 kD respectively).These data suggest the importance of how blockingand washing steps are performed, as well as the temperature of blot washing and antibody incubation.Hands free western blot processing using BlotCycler enables researchers to optimize shaking, washing,and antibody incubation condition during westernblot processing to eliminate nonspecific bands andgenerate high quality results.

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