Recently, I have faced an issue with my antibody not picking up my band of interest correctly. Previously, this antibody has worked with no issues at all (expected blot) and has produced beautiful clean bands, but some months ago, the same antibody started to blot really blotchy bands. Surprisingly, my beta-actin blots perfectly. I have reordered the same antibody but it still behaves the same way even though the lot numbers of the antibody are different.
I have tried running my gel slower and at a lower voltage, doing a 2h primary antibody incubation/1h secondary incubation, leaving the primary on overnight, using both ECL and West Femto and making new aliquots of my primary. None of these have worked in my favour.
Is there anything else that I can try?
Hello, the issue can come from the quality of your samples. the well keeping of samples is very important because with time and especially if not well preserved, the samples may lose their proteins content. The keeping of the antibody is also very important, so I hope you are doing this properly.
Hello Ruchi,
I disagree with Dr. Iskander Madhi, i don't think this is an issue of protein quality. I think its related to how you are incubating your membranes with antibodies. They must be kept at 4 degrees with continuous, gentle shaking (I am speculating here that you aren't keeping your membranes for antibody incubation on a shaker). Also, I would like to know the size of the protein of interest. If you are using a shaker, then it could be something else, cannot say for sure, until we get to know the protocol you are following.