New blot problems - (Jan/06/2009 )

In the last month I have come upon some Western Blot problems. While in the past I was getting strong protein bands with little non-specific binding, now I am seeing substantial background, hardly visible bands, and multiple non-specific reverse bands. The strongest reverse bands are my ladder bands. I have tried remixing most reagents (SDS Page buffer, Towbin, and TBS-T), I have repurchased my secondary antibody (I was sure this would fix the problem), and I have a new batch of precast gels. None of these things seem to have worked. I have also tried multiple different batches of detection reagents- ECL and ECL+. My primary antibody is fairly new (October), and it worked well when it first arrived. I don't think this is the problem. I have continued to be successful with mouse protein blots, which points to my mouse anti-human primary and goat anti-mouse secondary as the culprits. But, like I said, they are both fairly new.

Any ideas what this could be?

Interesting suggestion...

I block with a milk/TBS-T solution. I haven't tried adding blocking agent to my Ig solutions. Maybe I'll give this a try in the future.

The whole process seemed to work just fine but a month ago. I think I'm sort of trying to pinpoint and correct what has changed...

Normally reversed bands derive from too high concentrations of the second antibody. You may try to reduce the amount you are using now. Also always add blocking reagent at least to your first antibody, results are almost much better, less background. In the second it is also helpful, give it a try!

I'm definitely going to try blocking the primary. Thanks for this advice.

As to the protein volume... I have definitely heard of and experienced reversed banding when there is excess protein. But my plating and harvesting has not changed. The fact that the ladder is creating the strongest reverse bands leads me to believe that this is a reagent/antibody problem, not a protein volume issue.