My proteins appeared but actin not. May be I forgot to add the secondary antibody to the wash buffer or primary ab actin was expired. So I need to repeat the all antibodies with new gel run? I know the proteins are there and expressed in my sample but there was technical error so actin did not show.
My preference would be to start again with fresh blots. There are protocols for stripping blots that allow reprobing them. I only tried this once and wasn't satisfied with the result, but other people may have had a better experience.
you can start again the antibody incubation by stripping buffer. after stripping you should block again with the skim milk or serum bovine albumin and incubate again after several wash with TBST. At least due first incubation for over night in a roller at 4 degree.
Try to not repeat it for more than twice because the protein affinity will decrease it if you want to measure the quantity, it will not be reliable.
Good luck
Depending on your particular antibodies and the membrane stripping protocol, the stripping procedure might work and might be useless. Some Abs with very high affinity to your proteins might not be completely removed during that procedure, and that will affect the final results of your western blot. I'd suggest to repeat the SDS-PAGE and western blot again if you want to get reliable results.
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