My antibodies work beautifully and I get a nice difference in protein expression between tumor and control samples. My problem is, after I strip the membrane using harsh stripping protocol (@ Abcam using beta-mercaptoethanol) and wash thoroughly for 30 min under water and probe with beta-acting, I get no signal.
My PI has in the past, made it work and I don't know why it doesn't work for me. Can any one help me out.
I have re-used the primary Ab only once before and I was very very careful. Is there a possibility I over-washed the membrane?
If anyone can give me any insight on what to do next or what you think went wrong, it will be very helpful.