Protein quantification

Peter Pediaditakis pxpst2 at dlt.pitt.edu
Fri Mar 2 10:20:06 EST 2001


In article <3A9EC6DE.BB952FFF at acs.ucalgary.ca>,
 Jill Johnston <johnstoj at acs.ucalgary.ca> wrote:

> I need to quantify protein in SDS/DTT sample buffer.  Most of the assays
> I know of are incompatible with SDS and DTT.  Are there any out there
> that I could use?

I use a micro BCA protocol.  My sample is in 1-5 mM DTT with 1 % SDS.  I 
normally dilute it 10 fold and then use either 2 or 5 ul of sample in 
the assay.
Volume of reagents for assay
10 ul sample (2ul diluted protein standard + 8 ul lysis buffer -DTT 
w/SDS)
200 ul BCA mixture.

protein standards are between 0-10 ug ( I use a stock of 1 ug/ul , now 
You see why the above sample volume is fixed at 10 ul)

High concentration of DTT will mess with this assay but SDS is no 
problem.  Due to my dilutions, I do see much deviation from linearity.


for more info go to:
http://www.the-scientist.com/yr2000/oct/profile_001030.html


regards

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology

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