Bradford assay+high urea - (Oct/28/2009 )

I have protein solublised in 8M urea buffer, do you I can apply bradford assay for protein solublised in high conc. of urea?
if it is OK do I need to prepare the standards from the same buffer that my protein is soulube in( 8Murea)? or I can use H2O?

bio-rad says that their reagent is compatible with 6M urea. that's undiluted sample in the standard assay. if you dilute or use a small volume (relative to the reagent volume) then you should be okay with 8M.

it is always better, but not always necessary, to run your standards in the same buffer as your sample (we use a buffer blank because we may be running samples with more than one buffer).

I will use a 96 well and I will pipette 0,2,4...15 and 20 micL of BSA STANDARD solution and make up with ddH2O 20micL , and the sample is 20micL , and 200micL bradford reagent. Do you think the ratio between the reagent and the buffer is relatively small in this protocol?

the standard assay is 4:1 reagent:sample, yours is 10:1. it should be okay. try it.