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Reprobing PVDF membrane

Tori Brophy sibley at genetics.washington.edu
Fri Dec 1 12:44:51 EST 1995


sabah hussain <shussain at rvhmed.lan.mcgill.ca> wrote:
>Dear netters. I am looking for a protocol to strip primary-secondary antibody complex 
>from PVDF membrane  so as to reprobe PVDF membrane more than one time.
>I know the exitence of a such protocol for nitrocellulose membrane
> (Parek B., Analyt. Bioch. 1985). I appreciate any help in this matter. 
>
>

Pierce includes a stripping protocol with their SuperSignal enhanced 
chemiluminescence instruction sheet.  I've only used it a couple of times but it 
seems pretty harsh.  You might lose some protein from your blot.

1.  After exposure to film, put the blot in wash buffer.

2.  To strip, incubate the blot with stripping buffer (2% w/v SDS, 62.5 mM Tris-HCl 
pH 6.8, 100 mM B-mercaptoethanol) for 30 minutes at 50-70 C.  

3.  Wash the blot with wash buffer 2 times for 10 minutes each.

4.  Incubate the blot with SuperSignal and expose to film to determine if it's 
stripped enough.  Repeat if not sufficiently stripped.  

I tried using 0.5 X stripping buffer once and that worked fine.  It might not be so 
hard on your blot.  


Tori Brophy
torih at genetics.washington.edu





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