Western blot - Reversed polarity (Aug/04/2004 )

Hi,

I have been having a problem with Western blot for the Dopamine receptor 2 (D2). The background comes out black and the appropriate bands come out clear. The rainbow contains both clear and black bands and there are some dark (darker than the background) bands present in the sample lane. The D2 bands always come out strikingly clear. The problem is specific to this antibody; our lab has had no problems with the secondary antibody. The manufacturer insists that it is a development problem. However, our lab has also not had any problems with development of other WBs. Our initial thought was that the signal was too high. We have cut the sample amount in half and titrated from 1:800 to 1:10000 primary antibody concentrations. The manufacturer reccomends 1:800 and publications used 1:400. The result is the same, except for the 1:10000, where a strong background is still evident, but the white bands are faint. The 1:5000 did have a lighter background, but the bands were still very clear. Three different blocks have also been used (goat milk and serum and cow milk). I also thought that excess second antibody may be an issue. I rewashed the membrane for several hours and redeveloped with no decrease in background. I appreciate any help that can be offered.

Thanks,

Jason
University of Michigan

How long are you exposing the blot for? You might want to cut it down to just seconds...

I have exposed for as little as 1 sec. The problem is the same, only a little fainter. I may have some idea about the problem. Because of reactivity problems, only goat's milk or serum can be used as a block. BSA is known to give extremely high background. We always dilute our antibodies in a 2.5% BSA solution. I did several dot blots (protein directly onto the membrane, without gel separation) and ommitted BSA from the primary. The 1:10,000 looks pretty good, whereas the bands were still reversed when I did the standard Western at this dilution (containing BSA). Could someone tell me if this is possible: the antibody binds strongly to the antigen, but also reacts with BSA in the solution. Subesequently, the secondary antibdy is blocked from binding to the primary complex, but creates a high background everywhere else. This is the only way I can rationalize the presence of clear bands, with high background and some dark bands. I think I will take a few days off from Westerns and try again without BSA (perhaps using goat serum).

Omitted BSA from both primary and secondary dilution; problem solved!