1) Keep the membrane in methanol for 5 min.

2) Boil the membrane in 10 mM EDTA solution for 1 min (In microwave).

3) Block the membrane with 5% milk solution and probe it with antibody.

Ok ,thank you ,I will try.

This is what we do in our  lab:

For Mild stripping:

Buffer, 1 liter

15 g glycine

1 g SDS

10 ml Tween20.

-Adjust pH to 2.2

-Bring volume up to 1 L with ultrapure water.

-Membrane incubation

-Use a volume that will cover the membrane. Incubate at room temperature for 5-10 minutes.

-Discard buffer.

-5-10 minutes fresh stripping buffer.

-Discard buffer.

-10 minutes PBS

-10 minutes PBS

-5 minutes TBST

-5 minutes TBST

-Ready for blocking stage.

While for Harsh stripping

Prepare buffer and strip membranes under a fumehood.

Buffer, 0.1 liters

20 ml SDS 10%

12.5 ml Tris HCl pH 6.8 0.5M

67.5 ml ultra pure water

Add 0.8 ml ß-mercaptoethanol under the fumehood.

Cheers!

Hello  , 

Kindly follow links and read the protocols. I hope, they will help you at some extent. 

Regards,

Babu

http://www.bio-rad.com/en-bd/applications-technologies/stripping-reprobing

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679975/

exactly  Uday Munagala sir 

Hello ,

I have got a question concerning stripping with Immunoprecipitation western blots.

I did the IP-Westernblot with nitrocellulose membrane it works really nice, but my protein of interest have the equal band than the housekeeping gen.

I would like to strip my membrane..... I did it with nitrocellulose and PVDF membrane.

In Both cases, the protein was washed out of the membrane:

PVDF membrane with mild stripping buffer....the band was still there after checking the signal with ECL under chemiluminescence

With ß mercaptoethabol.... everything was gone!!!!

I used the mild and harsh stripping buffer which is recommended from thermofisher.

Maybe I should try the ß mercaptoethanol with 50°C for 20 minutes and nit 45 ??

Or I should use the Nitrocellulose membrane with the mild buffer , but only wash the membrane one with this buffer solution.

Have you got some ideas?? recommendations?

I just use TBST 1% HCL for no more than 10 minutes, and it works fine, I have no background of previous antibody and the signal of the antibodies tested after striping its o.k, I haven't strip more than 2 times, but for what I have read you might be able to do it at least 3 times.