western blotting using alkaline phosphatase
Posted 27 July 2004 - 04:25 AM
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.
Posted 30 July 2004 - 07:56 AM
Posted 30 July 2004 - 02:02 PM
Posted 02 August 2004 - 06:11 AM
Thanks for responding.
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?
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.
Posted 04 August 2004 - 08:46 AM
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?