Western Blot stripping - quick and easy protocol? - (May/23/2005 )
I am looking for a quick and easy way to strip a Western Blot and to repeat the detection with both primary and secondary antibodies. I usually blot onto PVDF and detect the primary antibodies with alkaline phosphatase-conjugated secondaries. The color reaction is with BCIP/NBT.
I have already found a protocol for the stripping procedure itself (biochem.wustl.edu/~pike/ methods/Western_Blot_Stripping.pdf). But I still have to get rid of the blue precipitate from the AP-reaction. I know from Southern Blots that I can wash this away with warm Dimethylformamid.
The question now is:
Can I combine this two steps in a save way, allowing a successfull second detection?
Does anybody here have experience with this, or maybe a suggestion for a better alternative which works with the BCIP/NBT-system?
that was discussed here for striping antibodies.
You can buy western stripping buffer from pierce, all you have to do is incubate your membrane with ~15ml for ~15min, wash and your away.