Protein Aggregation, Sample Boiling, help! - Western Blotting (May/20/2011 )

I believe I'm having a major protein aggregation issue with my whole cell lysates, but I can't pinpoint the cause. The thick prominent band in my 10% blot (examples pictured below) is a non-specific band. I am looking for pSmad2 which is around 60 kD, so this thick band runs right through it. I boil my samples for 5 min at 100C immediately after lysis and before loading into the gel. My DTT concentration is 150 mM. I have tried centrifuging before loading, extensive sample dilutions, and I've had no sign of improvement. What else can I do to break up this band?

Have you tried sonication of samples?

I take it these are not immunoprecipitations?

Try lowering the concentration of the protein in the lysate - use more lysis buffer or fewer cells.

cwiloby05 on Fri May 20 15:11:08 2011 said:

are you lysing the cells with sds sample buffer? if not then boiling without sds (and reducing agent, but not always) can lead to aggregation of proteins

is there residual cell culture medium with the cells when you lyse with sample buffer? you may be seeing albumin from the serum in the cell culture medium (it can show a depressed molecular weight when overloaded).

if so then you may want to wash your cells with serum-free medium a couple of times prior to lysis