Posted 27 January 2009 - 01:59 AM
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!
Posted 27 January 2009 - 05:56 AM
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
Posted 09 February 2009 - 11:14 PM
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...