BCA protein assay

[Guest jonas]

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!!

[daisy]

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

[m/z]

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.