MM yeast two hybrid - bradford measuring of yeast proteins - (Mar/15/2006 )
Hi,
can anyone help me with Bradford test to measure the protein obtained from yeast? I'm using the MM yeast2hybrid system3 from Clontech and have to do the western blot to confirm my baits, if my sequence is functional and gives the right protein. I did the protein extraction and have my proteins in cracking buffer. To measure the proteins I'm trying to use bradford reagent from Sigma. The problem is that I can't dissolve the BSA in the same buffer, in which I have my proteins from yeast! But I have to measure the standards in the same buffer, right? any idea?? thanks a lot!! 
you should be diluting your samples and standards with water prior to adding the bradford reagent. prepare your bsa in water. add sample buffer and bsa to the standard tubes and adjust to final volume with water. then add bradford reagent and read.
Thank you!
I tried to do like you wrote, but anyway the absorption is not the same when I compere measurement of BSA standards diluted in water and diluted in water with 1/10 cracking buffer (8M Urea, 5% SDS, Tris-HCl 40mM, EDTA 0,1 mM, bromophenol blue 0,4 mg/ml ). I think this buffer can prevent the Bradford test...
I tried to do like you wrote, but anyway the absorption is not the same when I compere measurement of BSA standards diluted in water and diluted in water with 1/10 cracking buffer (8M Urea, 5% SDS, Tris-HCl 40mM, EDTA 0,1 mM, bromophenol blue 0,4 mg/ml ). I think this buffer can prevent the Bradford test...
the absorbance with the buffer added should not be the same as without. that's the whole point of including the buffer (same volume as sample) with the standard. bromophenol blue, sds >0.1% and >6M urea (concentrations prior to addition of dye reagent) will interfere with the assay. so you are correct that your buffer may interfere.