membrane stripping methods - (Jan/19/2008 )
I am planning to strip membranes on which I did not get any bands for my samples (but recom. std. proteins showed up bands).Now I want to try out my primary antibodies at 4 o C (over night incubation) instead of 2 hr RT. Hence , in order to strip and re-probe, can anyone give me an efficient method with complete details of receipes for all the solutions etc. Also , i would like to know if the secondary and detection methods have to be the same as before stripping.
Stripping should not affect your re-probing efficiency, although it varies depending on your membrane (PVDF and nitrocellulose can handle stripping well). To prepare a stripping buffer, add: 0.2 M glycine, 0.1% SDS, 0.1% Tween 20, and adjust pH to 2.2 . Incubate your membrane with stripping buffer for 20 min (with shaking) 2 times. Then wash 2 times with 0.05% TBS-tween 10 min each. Your membrane is ready to go!
P.S. If you use PVDF membrane, make sure that you wet your membrane with methanol before adding stripping buffer. You should be able to strip the same membrane multiple times (I personally stripped membranes twice)