protein concentration - how to determine the protein concentration very accurately?? (Jan/27/2009 )

Hi!

I need to determine the concentration of my purified protein samples as accurately as possible.
So far I have tried A280, Bradford assay and loading of the proteins onto SDS-PAGE to determine the concentration by eye.
My problem is, although I try to work in triplicates and be as precise as possible, that Bradford gives me very different results from SDS-PAGE. This "very different" means that I get up to 3 times difference in concentration, and that these differences do not follow any rule. For the series of samples, for some I get bigger estimation by Bradford, for others by SDS-PAGE, and only sometimes (rarely) these numbers coincide.
Can somebody tell me if he/she had similar problems? In my institute the inaccuracy of Bradford seems quite common, but nobody yet told me how to solve it?
Maybe I should try to change the standard protein used in Bradford Assay from BSA to something else (I have read that Bradford can underestimate quantity of proteins low in Arg content - my has 3.8% Arg - is it low or not??)...
Or try to change Assay - has anybody some experience with for instance Lowry?

Thnx in advance, cause this is starting to drive me crazy!

Ana

Hello

Lowry is considered as the best, then BCA protein estimation (for most proteins I use BCS protein estimation kit from Pierce).

If my protein is impure, I use densitometry where i run BSa standards with my protein on teh gel and quantify teh band, thendraw a standard curve b/n the conc. and quantification results and get the conc of unknown protein from tehre.

Also, estimate in triplicates.

Hope its helps

TC

hey!

thnx for the avice. after i've tried A280, bradford, lowry and BCA - BCA was the best for me - i've got the best standard curve and finally got the protein bends to look more or less the same on SDS-PAGE. and it was very important to do everything in triplicates...

A