Lost protein from blot? - (Jun/30/2010 )

I probed my blot with antibody and got beautiful clear bands. I stripped with a commercial solution which my lab uses frequently with no problems. When I reprobed with a different antibody, which I have used successfully before, I got absolutely nothing. Completely clear film. All of my marker bands have disappeared also; these were getting lighter and lighter even through the washes in the initial process. Has this ever happened to anyone else? I'm using a nitrocellulose membrane, blocking is 5% milk in TBST (0.1% Tween).

how do you develop your membrane? I see you talking about film, so it must be chemiluminescence, correct? I don't think the problem is with blocking. Don't use Tween 20 next time. use normal TBS.