I have a bacterial mutant that does not express a protein of interest. So I preabsorb the Ab (homebrew, raised from rabbit) with lysate of that mutant and then tried to detect the protein of interest in wild type and overexpressing strain. However, I still get a lot of non-specific bands. I preabsorbed the Ab with lysate at 1:100 for 2hr at room temperature. Then I centrifuged ppt at 14,000 rpm for 30min and collect the supernatant for western blot. Is there any way to improve the performance of Ab? Thanks
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