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BCA Protein Assay Problems - (Jun/29/2009 )

Hello everybody,

I face certain problems with the BCA protein assay: I did some (first) testings with BSA standards that worked quite well. Using the same standard assay procedure with IgG also led to nice calibration curves.
Now I want to determine TNF quantities in presence of L-Cystein, which seems to be quite difficult: Ive been using defined L-Cystein add-ons to the TNF standards but instead of TNF-dependent calibration curves I got more or less a bad extinktion constant.

Additionally my TNF samples show higher extinctions than the described TNF/L-Cystein-calibration standards.
My TNF sample consists of 10mM Na2HPO4/NaH2PO4 buffer, 2mM EDTA, 30mM NaCl, pH6,7 buffer. So far I could not find any literature saying that one of these substances interfere with the BCA Protein Assay.

Does anybody have a clue how to solve one of my problems?



i'm not saying that this is your problem (it depends on how much sample you add to the assay) but, edta chelates copper in the bca assay.