Loading controls for Western blot - (Nov/25/2005 )

Hi, I'm running Western blots of protein extracts from rat smooth muscle tissues, and I'm having trouble finding a good loading control. I've used b-actin, but I get doublets in all the samples that I've used, even with the use of a fresh cocktail of protease inhibitors. Supposedly these doublets don't appear in culture, so is b-actin unsuitable for tissue extracts?

When I tried GAPDH, its expression was all over the place for the different samples- different rats, but same organs. Moreover, GAPDH expression did not correlate at all with protein determination by the Bradford assay.

Any suggestions on why I'm seeing these various issues? Is there a more suitable loading control that I should be using (e.g. tubulin)? Comments much appreciated.

well i'm working with p53 in number of various cell lines and it's a good one.

I have used beta-actin and GAPDH as loading control for human cells and found beta-actin is usually expressed low and is not a good control in terms of getting a nice picture. GAPDH is strongly expressed in most cells and its expression coorelates well with protein quantification. So we stick to GAPDH.