Accuracy of protein quantitation methods - (Dec/20/2006 )
Hi, can anybody explain me why I get completely different results of protein quantity of one sample measured with Bradford method or analyzed with densitometry (Quantity One software) on SDS-gel stained with Coomassie. There is approximately tenfold difference or even bigger. I use different standards, BSA for Bradford and Carbonic anhydrase for gel. I know it can influence result but still I think tenfold difference is too big. Thanks for your ideas
the bradford method uses coomassie blue g-250, gel stain is usually r-250. r-250 binds in a less specific manner than g-250 so will be stronger than g-250. also, absorbance maxima is different.
if you are using g-250 to stain your gel, the stain solution is under different conditions than the bradford. bradford reagent shifts from near red to blue, stain is sort of greenish-blue.
standard does make a big difference. bsa gives about twice the absorbance of that from igg. try using carbonic anhydrase for your bradford standard.
is your protein of interest similar to carbonic anhydrase? if not then you may want to find a standard that is similar to (or the same as) your protein for use as a standard.