I think my proteins on the PVDF membrane are gone, because there is nothing to detect anymore.
But on the other hand the membranes smell after a lot of washing procedures still of 2-ME. Maybe my proteins are there and the antibodies cannot work because of 2-ME traces?
What do you think about that?
If there is no protein anymore I have to repeat the experiment but if they are there and it is only the antibody problem how can I get rid of the 2-ME?
The Ponceau staining shows no results except of proteins >150kDa, but Ponceau on PVDF is never good.
I am a bit desperate to be honest.
Would be great if anybody has faced such a mistake. I cannot find anything about it in the internet...
Thanks in advance, Anja
10 fold means ? 1 M ?
the concentration of 2-ME in our stripping buffer is 100 mM
aside from that I don't think the excess of 2-ME would be a problem
but for example the concentration of SDS
Hello Erman, yes I mean 1m instead of 100mM. The concentration of SDS was not the problem, I added just beta Mercaptoethanol in a judge amounbt and could not detect anything afterwords.
Hi
might it be helpful drying the membranes at room temperature and then AB incubation ? Because 2-ME has a very short half life
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