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).
Thanks for all the suggestions!
@Kristýna Hrazdilová: The Proteins are all between 15-20 kDa so I use a 15 % SDS-Gel, but have also previously tried 10 % with similiar results for the Blotting.
@ Ianina Pokholenko: So the thing is, that in order to extract the Proteins from the Spore I cook them 2 hours at 95 °C with 0,1 M NaOH, 0,1 M NaCl, 0,1 M DTT and 1 % SDS bevore using them for either the Dot Blot or the Western Blot. I do not fully know, but guess that they are denatured from this treatment. If they are not, they will probably also not be in the SDS-Gel. The epitope we are trying to detect is a his-tag and we are using the penta-his antibody for the detection. But we may try a different antibody.
@Imran Khan: Yes I have stained the Gel after the transfer, and get almost empty Gels with a little bit residual proteins for the higher molecular weight (not the range of my protein though) and sometimes a middle molecular weight band, though not as strong as without transfer and also bigger in size as my proteins. The Proteins I want to detect are between 15-20 kDa and the same size as the one which I can detect. Nevertheless my Transfer Buffer contains 0,1 % SDS. But I have no problem with Overtransfer, as I see nothing on the second Blot if I use two membranes, even from the Protein, which I can detect. I have tried PVDF, Nitrocellulose and Nylon, where Nylon did not work at all, Nitrocellulose worse than PVDF which had the best results. But I might test different blocking reagents (Milk and BSA, or do you have other suggestions?)
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