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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?

Thanks,
Kataya

-Kataya-

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.

-mdfenko-