Precise protein concentration

Biology jsullivan at moon.sba.dal.ca
Wed Jan 6 15:08:08 EST 1999


Hi
I am not sure if you have already done this or not, but if the
correlation between the concentration and Abs. is not linear, try to
dilute the protein sample before adding it to the 2 ml reagent in BCA
method. dilution as 1/2  or 1/4 may help. then add 100 ul of diluted
sample to 2 ml reagent.

Mahmood

Lena Zaitseva wrote:

>       Hi,
>      We purified a membrane protein in the presence of non-ionic
> detergent. Now we need to calculate precise protein concentration. It
> appeared to be a real problem.
>      Bradford method doesn't work at all. BCA method looks better, but
> with BSA as control it doesn't give us reliable results (there is no
> linear dependence of OD from concentration of our protein). For
> UV-method it would be nice to unfold  protein to get the maximum
> absorbency at 280 nm. However, guanidine-HCl and SDS lead to the
> protein aggregation. We tried also make quantitative amino-acid
> analysis of the protein, but  presence of high salt concentration (1.5
> M) and glycerol in the sample buffer is a problem in that case.
>      Do you have any idea about that?
>      Thanks for your suggestions.
>      Lena.


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