I am having Problems Blotting specific Proteins on PVDF and Nitrocellulose from the Spore Coat of B. subtilis. The proteins are visible in the Coomassie stain and are loaded as a mixture of all proteins as well as the purified version. It is not a general Blotting issue, as I can detect one of the Proteins on the same Blot. It also does not seem to be a Antibody problem, as I have no problem in detecting them in a Dot Blot. There are a few things I will try in the future, but I wanted to know, of there is some specific advice, or if someone already had such a problem. I will try to equilibrate the Gel in MeOH for the PVDF membrane, I will use a stack of 2 membranes to exclude overtransfer, I will stain with Ponceau to see how the lanes look like (even though I already stained the gel after transfer and it is more or less emptly). Any other suggestions?
Based on the size of the protein you can also try less concentrated gel and/or vary the time of blot
In case if your antibody detects the conformational epitope you will have the situation that it detects your protein in the dot-blot and don't detect it on western'blot after the standard SDS-PAGE under denaturing conditions. So firstly of course you have to be sure in effective transfer and then you have to be sure that the antibody is specific to the linear epitope (if it is monoclonal of course, because for polyclonal antibodies it is less common situation).
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