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Reprobing a PVDF membrane which has gone dry - (Jul/20/2012 )

Hello everyone,

I have recently done my first western blots and now I would like to reprobe one of them. I have used a PVDF membrane for this blot and I used a secondary antibody coupled to alkaline phosphatase. I would now like to look at the same protein on the blot using a fluorescent antibody instead (more quantitative than a method relying on precipitation as I understand). But now I have three questions:

1) Can I reprobe a protein which has already been visualised with alkaline phosphatase or will the product precipitate which allows you to see the band inhibit any further antibodies binding?

2) Which stripping method is recommended for PVDF membranes (would glycine pH 2 for example be okay?can SDS be used for stripping?)

3) I actually allowed the membrane to go dry after the seeing my first result because I thought I would not have to reprobe it. Is it possible to rehydrate the membrane with ethanol/methanol, strip and reprobe, or is the fact that it is completely dry a problem?

Thank you!


You can rehydrate and you shouldn't need to strip.