We currently use restore stripping buffer https://www.thermofisher.com/order/catalog/product/21059[1] which we usually incubate for 10-15 minutes to strip. For lower molecular weight proteins, we usually don't even bother because we know it'll just pull it off.
If I incubated it for like 5 minutes, would that suffice? Does anyone have experience with this. We have two proteins at 27 and 24kDa, same species so its tough.
Kevin: The best strategy is to use freshly transferred membrane for detection only once for LMW proteins. In my personal experience, the stripping buffer is OK to use for abundant and >50 kD proteins such as transferrin and fetuin in serum. Someone used peroxide (H2O2) to "kill" HRP in the prior blotting. However, I found that it sometimes destroy (oxydize) key amino acids such as methionine in the Ag epitop for Ab recognition. My adviser once said: "science is not based on convenience".
you probably mean sodim azide to kill HRP. (this is the way I do when stripping is not convenient/necessary) ..H2O2 is a component of the "ECL" reagent!
Didier: Na azide only reversibly inhibits but unable to inactivate HRP. So someone used H2O2 (up to 30%!) to inactivate/strip HRP (Anal Biochem 393: 129–131, 2009; http://www.sciencedirect.com/science/article/pii/S0003269709004011 ).
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