when using PVDF membranes do I have to activate my membrane before and after transfer or only before? I stained with Ponceau and I barely see bands. After revealing the membrane I get no signal.
I suggested that I had lack of protein loaded but I loaded 30ug.
I ran an extra gel in parallel to stain with coomassie and saw lots of protein bands.
I saw some comments saying that you have to activate your membrane with methanol prior to transfer (and I've always done it) but also after transfer (before ponceau staining).
After barely seeing bands in my "stained" membrane I washed it in water and after with TBST and bloqued it right away.
Do you think I should have wash the membrane in methanol before staining or not?
Hi Claudia, from personal experience, activating the membrane once (in 100% methanol for ~30 seconds) before transfer is sufficient (ensure it is fully submerged and part of it doesn't get exposed to air when the membrane floats to the surface. Also, don't expose it to water prior to activation). After activation, remember to incubate the membrane in transfer buffer for ~7 minutes (according to invitrogen/Life protocols) after the methanol and before the transfer (again without it floating to the surface and drying out). After transfer, going directly to Ponceau should work. Typically, I avoid washing in water, PBST or blocking buffer should be better if not just as good. Regarding activating after transfer, I've never heard it done or seen it done. I'd be interested to read responses from others who have performed this step.
Hi Claudia, from personal experience, activating the membrane once (in 100% methanol for ~30 seconds) before transfer is sufficient (ensure it is fully submerged and part of it doesn't get exposed to air when the membrane floats to the surface. Also, don't expose it to water prior to activation). After activation, remember to incubate the membrane in transfer buffer for ~7 minutes (according to invitrogen/Life protocols) after the methanol and before the transfer (again without it floating to the surface and drying out). After transfer, going directly to Ponceau should work. Typically, I avoid washing in water, PBST or blocking buffer should be better if not just as good. Regarding activating after transfer, I've never heard it done or seen it done. I'd be interested to read responses from others who have performed this step.
To wet/hydrate the membrane before protein transfer with PVDF if very imortant.
Once should be enough, but once wet in methonal keep in transfer buffer, without allowing to dry again.
PVDF membrane is very hydrophobic, meaning that aqueous buffers, such as the Western transfer buffers or deionized water, will not penetrate the membrane. This results in poor transfer and significantly reduced binding of proteins. A short rinse (15-30 seconds) in methanol (or other 100% alcohol (ethanol or isopropanol)) prior to Western transfer will "hydrate" the membrane and allow improved transfer and protein binding.
https://www.researchgate.net/post/How_long_should_PVDF_membrane_soak_in_the_methanol_solution_and_why
https://info.gbiosciences.com/blog/pvdf-methanol-soak
Best Wishes!
Hi Cláudia,
First, put the PVDF membrane into methanol for 1 minute. Then, dip it into the transfer buffer until use. Immediately after the transfer of sample protein into the PVDF membrane, follow the blocking step. After the blocking is done, wash the PVDF membrane once with milli Q (MQ) water. Then, go for incubation with antibody. Hope that you will get the band. All the best.
To answer to your question, after the transfer is not necessary to use methanol again. However, some protocols recommend to dry the membrane after the transfer and before blocking, which can be done with air-dry (Physical) or methanol (chemical) to achieve permanent fixation of proteins to PVDF.
Obviously, either way, once the membrane is dried you need to re-wet and re-activate it. In summary:
a) (physical) Air-dry the membrane for 2h
b) (chemical) Soak in 100% MetOH for 10 secs and leave it to dry for 15 min.
c) Re-activate the membrane before blocking/probing (by soaking in 100% MetOH for 10 secs, ddH20 for 10 secs and TBST for 10 secs).
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