BCA-Protein-Quantification: Time Drive?

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sun Aug 9 19:05:35 EST 1998


In article <35cab1d3.1944564 at news.fu-berlin.de>, 
deleteme_cpeter at zedat.fu-berlin.de wrote:
>Hi all!
>
>I use the bicinchoninic-acid-assay in a 96well format to
>determine/estimate protein concentrations. I always have a
>standard-dilution-row on every plate.
>
>When I tried to determine the concentration of a simple yeast-extract,
>I noted the following:
>
>- the slope of the calibration-line (mg as a function of A550)
>decreases with time (15min to 3hrs)
>
>- the concentration of my extract also decreases with time!!!!
>(calculated with corresponding slope!)
>
>As the assay is designed to be read after 30min and 30degrees C - when
>do I get the _correct_ concentration?????????????????

Anywhere it is convenient for you! That's what calibration curve is for. 
I, for example, to achive higher sensitivity, do the following: 1) put twice 
recommended copper (Pierce's BCA kit), 2) leave samples
for 3 hours at 50C. Works like a charm. Quite frankly, I have difficult time 
understanding how and where the color that forms in BCA reaction can 
disappear with time.

        - Dima





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