Protein measurements in 2% Triton X samples - (Jan/11/2012 )
do any of you know what techniques can be used to determine protein quantity in cell lysates that contain 2% triton x. I have lysed my cells in lysis buffer containing 2% triton, because I am looking at a membrane bound protein (LC3) by WB.
Bradford etc. reagents are reacting very! good with Triton X, and the reagent gets saturated very fast by Triton.
the bca protein assay is compatible with up to 5% triton x-100 (1% other tritons)
I have observed similar interference using a 2% Triton x-100 lysis buffer with Bradford assay.
Because triton reacts with coomassie blue (Bradford reagent), I ran a serial dilution of triton x-100 while keeping the rest of the buffer the same (no protein). The serial dilution was the following:
2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.0156%, and 0% triton x-100 (in HBSS).
I found that loss of interference by triton x occurred somewhere near 0.0625%, which could explain why many lysis buffer recipes only use 0.05% triton x (presumably because their designers were using the Bradford assay)
Thus if you find sufficient yield with as low ad 0.05% triton, the Bradford is a suitable quantification technique.