I'm experiencing quite some substantial problems with the BCA protein assay. I wish to determine the concentration of a his-tagged protein which have been eluted from a resin coloumn with TBS containing 5 mM EDTA in four fractions. When i meassure OD600 after incubation with working reagent the signal is rather high in the first fraction and absent in the latter three. However, when I run a SDS-PAGE and comassie stain it i see absolutely no protein in fraction 1, a nice band in fraction 2, and nice but weaker bands in fractions 3 & 4. Can anyone help me? Is it possible that stripping the resin with EDTA washes some compund, such as cobalt ions, which screws up the assay? please help!!

Hi Jonas...this link will give you a better forum to answer your question.....http://micro.nwfsc.noaa.gov/forums/..I don't know enough about protein chemistry to answer but if it was DNA, PCR, RNA, etc. I'd be right in there...good luck and that really is an excellent site

Actually, I can not give you a clear cut answer to this problem. However, I would say that the SDS-PAGE results are much more specific to proteins than the BCA assay. But you could try to do a blind test - i.e. elute the resin column without loading any sample and perform a BCA protein assay of the fractions.