I'm trying to evaluate the effect of virus infection on MHC class I expression, and am using GAPDH as my housekeeping gene, which should be showing standard levels of expression across my samples. I've been performing TCA precipitation since my cell lysates are radiolabelled and I wanted to reduce the amount of 35S going into the Western. However, even in replicates of my mock-treated samples, I'm getting huge variation in GAPDH expression.

We thought this might be due to losing some of the pellet during washes while precipitating, so my supervisor suggested precipitating onto Whatman paper to reduce possible losses during washing. This seems to work well, but we can't figure out how to get the protein back out of the paper again! I boiled it at 96 degrees for 10 minutes, but my gel was completely blank.

My loading buffer was still yellow when I loaded it, which I realise could be causing some of the problem, and so I'll be using TRIS to get it back to blue before loading next time.

But I still don't think that would account for a total lack of protein in my gels.

Does anyone have any tips for keeping your total protein as constant as possible while precipitating and Western blotting, either with or without the Whatman paper step?