We are running a western blot to check the levels of some proteins and using beta-actin to normalize the levels of our target proteins. We did the target proteins and were able to get good bands for our 4 samples, however when we incubated with a conjugated beta-actin (incubation overnight) only one band showed. We have been using Thermo Scientific Restore PLUS Western Blot Stripping Buffer to strip the blot in between antibodies. We blocked the blot with 5% milk and used 5%BSA for antibody dilutions, incubation was at 4C overnight for all of them.
Why are we only getting a signal for 1 sample for beta-actin even though we got good signal/bands with our target proteins which are much lower abundance?
This looks like bad protein transfer. Check the transfer buffer and especially make sure that the membrane firmly sticks to the gel and there are no bubbles between. If the buffer is standard (ie, containing methanol/ethanol), make sure it is properly stirred and cooled before use (overnight in the cold room). Otherwise the air bubbles come out during the transfer and may interfere. This often happens locally, so you may get some bands ok, but some smearing, like you say.
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