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.
torih at genetics.washington.edu