Hi, I'm still new to running western blots and I'm running into a problem with my stripping. The very first time I stripped my membrane (nitrocellulose), I incubated it at 50-70C in a buffer composed of 20 ml SDS 10%, 12.5 ml Tris HCl pH 6.8 0.5M, 67.5 ml ultra pure water, and 0.8 ml ß-mercaptoethanol for 30 mins in a small oven. I then rinsed it under running water for 1 hour, rinsed it 5x with TBST, and blocked it overnight at 4C. When I reprobed with my new primary antibody, I got a good signal exactly where I expected.
The oven is a bit shoddy, though, and I wanted to have more control over the temperature, so I tried my next strip (different membrane) by floating it in a water bath at 50C for 45 mins. After that, I had no signal from my old antibody or my new antibody. I thought perhaps something about the wet heat and longer time period had changed my stripping conditions and ruined my antigen, so I switched back to the oven at 50-70C for 30 minutes with my next membrane. And I still have no signal from either my old antibody (the one I was trying to strip) or my new antibody that I did my second incubation with.
I guess I just had an amazing case of beginners luck, because I cannot get a signal with my stripped membranes anymore! My plan for the next membrane is to incubate with the second antibody first to make sure it is still usable (to ensure it's the stripping that is giving me problems, not the antibody). If that checks out, I'm thinking of trying a more mild stripping buffer:
15 g glycine