western blotting using alkaline phosphatase - (Jul/27/2004 )
I am trying to detect FLAG fusion protein from plant fungus using Bio-Rad chemiluminescent protein detection system. My primary antibody is mouse anti-FLAG-antibody and secondary antibody is alkaline phosphatase conjugated anti-mouse antibody.
I am having a trouble because my negative control also gave me a signal as well as positive control (FLAG conjugated protein from Sigma) and samples. The protein samples were in 3M Urea buffer and denatured by boiling for 5 min, which means I guess there should be no active alkaline phosphatase from my samples that can give me false signals.
But I am not sure about this, so if anybody has some experience, please give me some advice. Thanks.
proteins can renature after your've transfered them to membrane. You could boil your membrane for 2min before you put your 2ndary. This might destroy your AP on your membrane.
When you say you get "a signal" in your negative does that mean you geta band at the same size as the test lane? Or is it just some background bands? Are you worried about random AP activity because this fungus has a lot of endogenous AP expression?
Thanks for responding.
I have only one band in negative control and test lane with the same size. Since I have no other band except that one, I think probably, the band is AP from my fungus and I could not detect FLAG-fusion protein because I lost the FLAG protein during protein extraction.
I guess doing chemiluminescence to another blot NOT probed with any antibodies will tell you if it is endogenous AP activity. If it is not endogenous activity then it is probably simply nonspecific binding of either your primary or secondary antibody to a fungal protein. In that case you can either live with it, or try to find other antibody combinations or wash conditions that might eliminate it.
You say you may have lost the protein in an extraction, can you detect your fusion protein on a blot of whole fungus lysate? I am assuming the spurious band is markedly differnt size than the expected size of your fusion protein, right?