Inconsistent stripping - (Oct/22/2014 )

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

Note - I'm really really not an advocate of stripping, as it is fairly common that stripping is inconsistent across membranes, and if you don't know how it affects your epitope(s) then how can you say that the results are consistent. If your proteins are sufficiently different in size, then just probe the blot with the second antibody and go from there. If they are close in size, you can block the action of HRP (assuming you are using it) with azide (test the membrane to show no signal after this), before proceeding to the next antibody.

My first thought was that it could be an antibody problem - so your testing the antibody is a good idea.

Humidity is a big player in these things, so incubating in a water bath could have some effect that you wouldn't get in an oven/incubator. However, if your membrane is submerged, this shouldn't play too much of a role.

My proteins of interest are close to each other in size, so I guess the sodium azide route would be my next best bet.

Is it alright to use the sodium azide to block my first primary antibody's chemiluminescence if my second primary antibody is from the same species (e.g. both of my primary antibodies are mouse anti-chicken and use the same HRP-conjugated secondary antibody)?

the azide will kill the hrp on the secondary antibody. then you start with the second primary. by the time you add the secondary the azide will be gone and won't affect the hrp on the fresh secondary.