If the protein you want to detect is sufficiently different from the one you probed for in the past, do not strip! You can always strip both signals later. Even the best stripping decreases the quality of the blot and loses some protein.

Re-wetting in methanol is a MUST. PVDF does not absorb aqueous buffers uniformly unless pre-wet. You may or may not end up with ugly splodges all over a beautiful blot! I tend to re-wet for a few seconds (the colour goes uniformly greyish), rinse in your wash buffer of choice (TBST being mine) and off you go. Re-blocking may be a good idea, but I have skipped it in the past when using good antibodies. If you don't strip, the blocking protein should still be there.

Good luck!

Hi Md. Atiqur Rahman,

You can try with the following protocol:

1. Re-hydrate in MeOH briefly (10 seconds), then rinse with water, then wash in TBST several times.

2. Then strip with stripping buffer for just 5 minutes, wash extensively in TBST

3. Re-block, apply primary Ab, and then wash and apply secondary Ab.

I think the following links can help you more

http://www.med.umn.edu/starrlab/prod/groups/med/@pub/@med/@starrlab/documents/content/med_content_370490.html

http://www.cellsignal.com/contents/resources-protocols/western-blot-reprobing-protocol/western-reprobing

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6218.pdf

http://www.abcam.com/ps/pdf/protocols/Stripping%20for%20reprobing.pdf

http://web.mnstate.edu/provost/Reprobe_StrippingProtocol.pdf

Good luck !!

Rehydrate in blotting buffer or PBST/TBST for half an hour with some changes (otherwise the salt concentrations might be too high), then re-block and add you antibody solution as usual. Keep in mind, that you don't want to bind proteins to the membrane as during transfer but to bind antibodies to the proteins that are already on the membrane. Therefore you don't have to activate the membrane again with methanol as it's necessary for PVDF for the transfer.

There are 2 different protocols and stripping buffers that you can use. in any case you need to rehydrate the membrane (just PBS or TBS) 5 min.

Protocol 1 (it smells, so you have to do everything in the hood):

30ml of water based buffer containing 6ml of 10% SDS, 1.875 ml of 1M Tris-HCl pH6.8, and 233μl of β-mercapto-ethanol was used per membrane. The procedure was carried out in a fume hood. The buffer was heated to 55C and poured over a membrane. The temperature was kept constant for 30min (the membrane was placed in a carrier in a water bath). Then the membrane was washed 4-5 times with TBS-T, after this comes blocking and the rest of immunodetection.

Protocol 2 (much better- and required if you use LICOR detection system)

Membranes were placed in a stripping buffer (25mM glycine buffer pH 2 and 2% SDS) for 15 min at room temperature. Then the membranes were placed in fresh stripping buffer for another 15 min. Following two TBS washes (15 min each) the membranes were placed inBlocking buffer for 1h, and re-probed with appropriate antibody.

Hope this helps.

If the protein you want to detect is sufficiently different from the one you probed for in the past, do not strip! You can always strip both signals later. Even the best stripping decreases the quality of the blot and loses some protein.

Re-wetting in methanol is a MUST. PVDF does not absorb aqueous buffers uniformly unless pre-wet. You may or may not end up with ugly splodges all over a beautiful blot! I tend to re-wet for a few seconds (the colour goes uniformly greyish), rinse in your wash buffer of choice (TBST being mine) and off you go. Re-blocking may be a good idea, but I have skipped it in the past when using good antibodies. If you don't strip, the blocking protein should still be there.

Good luck!

Than you very much to you all for your helpful advice.

Dear Afrin And Anna,

Could you please advice me what percentage of methanol should I use to re-wet the PVDF membrane. Thank you very much. 

Thanks for your reply & quest. Re-wet in methanol is must because of the hydrophobicity of PVDF membrane but some protein loss may occur. Re-wet the dry blot in 100% methanol for few second(10-15 sec) before placing in blocking. Some people also suggest to use 50% or 20% Methanol for pre-wet.  

Dear Afrin, Thank you very much for your suggestion. Could you please tell me why protein loss may occur and how I can prevent protein loss. Thanks once again.

The loss of protein on membrane caused due to stripping buffer which contain SDS detergent. The time & temperature for stripping are also important. You can try with mild stripping buffer in place of harsh or use some commercial stripping buffer which may reduce the loss of your target protein like "Restore™ Western Blot Stripping Buffer" from Thermo Scientific or NewBlot™ Stripping Buffers.

More importantly do not strip your membrane if it is not necessary.

Hope it will help you to get rid of your problem because I am not experienced enough to help. Best of luck for your research.

Dear Afrin, thank you very much for your advice. Actually, I am using the Restore™ Plus Western Blot Stripping Buffer. Wish you all the best as well.

I concur with Afrin - re-wet briefly in 100% methanol (no protein loss), and don't strip if you don't have to.

Dear Anna, thank you very much for your suggestion.

Hello. I know that my intervention is late but I just wonder why all of you use methanol to reactivate the membrane. It can be well done with absolute ethanol, it's less dangerous for your health and you don't have to use it under aspiration.

I think there is a good explaination but i am missing it.

Thank you

Dieynaba Ndiaye

Old but I'll respond because I stumbled upon this thread -- absolute ethanol is fine to use but methanol might be the preferred choice because:

A.) Higher purity. Reagent grade "pure" ethanol can still contain several solvents and impurities that can ruin your transfer.

B.) Ethanol-water mixtures are rather unique leading to tightly clustered H20-EtOH molecules that makes the solution more viscous. The wash steps following alcohol activation require a longer period of time to allow the EtOH to diffuse out of the membrane. Transferring prematurely from washing out all of the EtOH can also cause transfer issues.