I do Western Blot with PVDF membrane. Usually I can use Anti bodies diluted in Milk and BSA as long as I strip the membrane inbetween. I accidently didn't strip and now the membrane doesn't work anymore, any way I could "save" it? I tried stripping and reactivating with Methanol. I would really need to be able to do the load control at the very least...
Hi, can you elaborate a bit, how is the membrane "not working anymore"? what do you use for stripping? And did I get that right that you used methanol on a membrane that already contained your proteins and Ab from previous staining?
I do the same protocole as before but there are no bands anymore, no matter how long I let it developp (I have been up to 15 minutes). I tested the antibodies on other membranes, they are still working normally (normal developping time: 2 seconds for the load control antibody that I would really like to see to be able to analyze what I did befor, up to 30 seconds for the other antibodies)...
might be that your stripping was too harsh, what do you use for stripping?
If you haven't tried it already you can try to amplify your signal using ECL femto.
have you blocked your membrane again after stripping?
If you want to make an idea for yourself what the results are, you can always use a non-specific signal (or several) from your previous staining (if any) to normalize the result. It's not usable in a publication, as you never know if your treatment could affect this randomly selected signal. But chances are good it doesn't, so you can always measure it for yourself, and see if it is worth making the same experiment again.
The stripping is made with a mix of Tris-HCl, beta-mercaptoethanol, SDS and water. Usually I strip for 20 minutes at 55°C. At the start I looked on another membrane how many times I could strip-reinicubate with the same primary antibody before it modifies the signal and even after 10 times the signal strenght was basically the same (I checked after the strippings that the signal was all gone of course).
I always reblock the membrane after the stripping.
I can't use non specific signal as I have none (The histones are extracted very specifically from the cells)
Thanks for taking the time to answer
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