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Problems with stripping a western blot
Posted by: hyperion79 (IP Hidden, New member, 1)
Date: January 23, 2005 06:41PM
Hi, I have tried a very similar stripping protocol to the one described in "A step by step protocol on western blot with detailed description on reagent preparation, sample lysis, protein quantitation, gel casting, transfer, blot and stripping. (Jonathan Flint lab)"
However, reprobing reveals only faint traces when overexposed (greater than 10 minutes). The only difference I can see between the two protocols is I did not prewarm the strippping solution prior to incubation with the membrane. I suspect that either the stripping protocol is too harsh, and is removing the antigen as well as original antibody, or perhaps is incomplete? Does anyone have any suggestions as to why this may be happening? Many thanks Tricia Lusby Research Assistant Queensland Institute of Medical Research Brisbane, Australia.
Re: Problems with stripping a western blot
Posted by: huangz123 (IP Hidden, Junior member, 19)
Date: January 23, 2005 07:00PM
Try to strip your membrane in the room temperature for a shorter time. Make fresh stripping buffer. You can always increase the incubation time and temperature if the background is high. PVDF membrane is recommended if you will strip/reprobe for several times since it is more resistant to chemical and physical harsh conditions. Good luck!
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