My recent Western blotting has been unsuccessful. I've been using the CDKN2A/p16INK4a Antibody (YA3494) and p21 Antibody, but the results always show multiple bands, and the bands appear faint and unresolved. Why?
My experimental conditions: reducing loading, 5% BSA blocking, centrifugation for 15 minutes, reduced sample volume (approximately 25%), and transfer at 300 mA for 30 minutes.
Marine Ma , both antibodies should produce a single band (verified by the MCE validation team).
However, your experimental results reveal two major issues: numerous nonspecific bands with strong signals, and a very weak signal for the target band. This is likely due to excessive sample loading, leading to nonspecific recognition. Furthermore, there may be impurities in the sample. Here are two optimization suggestions:
1. Reduce the sample loading volume to around 25 μg;
2. Add a centrifugation step during sample preparation to remove impurities. Set the centrifugation conditions at 13,000 rpm for 15 minutes, three times.
The answer to this question comes from MCE Technical Support.
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