Plant protein quantitation - from crude extract (May/09/2006 )
I am really having trouble determining the protein concentration of crude extract. I m not getting the desired result. I'm using b-mercapto ethanol along with PMSF in phosphate buffer of pH 7 to homogenise the sample. The resulting crude extract has to be quantitated for protein content.
I took different dilutions of the protein samples and did Bradford assay but am getting different color from the standard BSA in reaction with Bradford reagent. With BSA, I get a blue color but with my sample I get a parrot green color. I am thinking the PMSF and BME are interfering so is there anyway I can get a better result. How can I account for these two compounds while performing Bradford assay?
is it possible to dialyze your extract to get rid of that stuff? or, dilute until it's no longer a factor?
you can prepare your standards in the same buffer as the extract. then all of your samples will be parrot green.
or you could use a different method for determining protein, one that is not as sensitive to 2-me and pmsf (eg-bca, lowry or biuret, depending on expected protein concentration). you will still need to blank for buffer.