Loading problems - please help

[kforema]

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!
Kim

[lyrachang]

1. Since your spectrometer was broken, it is likely that your protein conc. determination is off. Why don't you compare the results of both BCA assay and Bradford assay. Remember to do several dilution of your sample to get more accurate numbers.

2. You can increase the conc. of your loaded cell lysate.

3. Maybe its your Ab is going bad. Why don't you use higher antibody concentration and see if you can detect the protein of interest.