Alternatives to Bradford - Bradford disturbed by Lysis Buffer (Oct/15/2007 )

Hi,

I have a problem as follows:

I'm trying to quantify protein lysates for Western Blots. Unfortunately, components in the lysis buffer (Proteinase Inhibitor, NaF, NaVO4, ...) disturb the Bradford so that I even get strong absorption when there is no protein in (just lysis buffer + Bradford reagent)

Has anyone had the same problems...I already tried to dilute my samples further, no good results.
Of course I know that there are other ways to measure protein concentrations, like BCA, but I wanted to ask if someone had the same problem before and has a good solution to it.
I also tried to measure the absorbance directly at 280nm without any reagent, just diluted in water, but the lysis buffer alone gave a strong absorption again, so that I could not measure the amount of protein in my lysates.

Thanks for all your replies!

Stephan

the problem is due to detergents (triton X-100, NP-40...) and reductors (DTT beta-mercaptoethanol...) and SDS.
Have a look on biorad catalogues, there are kits for quantifying proteins in buffers containing detergents and/or reductors . I don't remember the name right now, but it works fine.

I'll second the BCA assay as a solution, and you don't even need the reducing agent compatible one if your lysis buffer doesn't contain over 1mM DTT, etc. Here's a link to more info:

http://www.piercenet.com/products/browse.c...;WT.mc_id=forum