Hi, I am pretty new doing WBs and I would like to reprove one of my PVDF membranes. I read that there are several possibilities but I do not know which is the better, or the advantage/disadvantage of each of them. I read about:
1) Acidic Method (25mM glycine-HCl pH2.0, 1% SDS)
2) Detergent Method (2%SDS prepared in Tris, 100mM mercaptoEtOH and maintained at 50 degrees)
3) Commercial options like the Restore WB stripping buffer from Thermo (Prod 21059). I think that this one is expensive, but...
I would really appreciate any advice about this topic. Thanks in advance!
The first one!! La he usado y funciona, aunque si tienes bandas muy cercanas, puedes llegar a confundirte. Algo mas rapido es usar 10ul de 10% sodium azyde en 10 ml de TBST, es un poco mas risky porque es sodium azyde, pero mas rapido....
Hi ! I used to strip my membrane with a wash of 15 min of citric acid at 0.1M (pH 2) after 3 wash with PBS-Tween. To be sure that the strip works, you should do a revelation with secondary antibody just after the stripping step. After stripping, I rinse my membrane 3 times (5 min) with PBS-T BSA 0.1% then put the new antibody solution. Good luck !
Hi Javier.
I tested many methods with different results. The best in my opinion is the commercial method. We use a striping buffer from carl roth, which doesn´t cost so much and we always have nice results.
As Blaise said you should always check if the strip worked. I do so by choosing a "control line", where I only apply the secondary ab, no primary.
In the second immuno reaction to be on the safe site I always choose to label proteins which don´t have the same molecular weight as the proteins I labeled in the first immuno.
Greetings
Please try abcam protocols for both mild and harsh stripping they work very nicely. good luck
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