I am having a terrible problem with loading western blots.
I have substantial experience with Westerns and previously had no difficulties with even loading (I use tubulin as a loading control, but have also used actin). Now, both myself and a technician in the lab (little experience) have been having problems so I don't think it is a technical issue, but some sort of reagent or protocol issue.
We use a RIPA buffer plus protease inhibitors for protein extraction and a bradford assay (BioRad) for protein determination. We are doing a 1:500 dilution so I thought the detergent levels would be low enough to avoid interference. I use to use a spec for reading absorbances, but our machine broke and we switched to a Polarstar Omega microplate reader that can do absorbance. I have used both linear regression and polynomial regression to calculate the concentration from the standard curve, but neither has solved the problem.
This is a serious problem. I can only get 1 out of every 5 or 6 Westerns to have even enough loading to be acceptable, and the rest are garbage. We are using NuPage precast gels and buffers, which have never been a problem in the past. I have racked my brain for answers, but can not seem to pinpoint the problem.
Any suggestions would be greatly appreciated!