BCA Protein Assay and Detergents!!

Jeffrey W. Chisholm JWChish at ac.dal.ca
Tue May 2 20:10:26 EST 1995


Somebody recently posted that up to 1% SDS could be added to the BCA 
protein system for the measurement of lipid rich protein samples.  As I 
work on lipoproteins and was in the process of switching to the BCA 
system from one of the modified Lowry methods (With SDS or Deoxycholate), 
this was of interest to me.

However, in my hands, 1% SDS in the BCA assay yielded significantly lower 
protein values in VLDL, LDL and other lipid rich particles as compared 
with the plain BCA protocol.  In addition, when using the micro BCA 
method (Pierce) with microtiter plates, the SDS was prone to 
precipitation with samples containing high salt.  As a result, I tried 
the BCA assay adding 1%, .5%  and 0.25% Deoxycholate as a solubilizing 
agent.  Overall, 0.25% appears to work best, yielding a std. curve (BSA) 
identical to the plain BCA protocol and giving significantly larger 
protein mass values in lipid rich particles and comparable values in 
lipid depleted samples.  1% and 0.5% DOC yielded protein masses equal to 
or less than the plain BCA kit. From now on, I will be using the BCA 
protocol with 0.25% DOC for regular determinations of protein in lipid 
rich samples.  Incidently, 0.25% DOC was the concentration used in our 
DOC modified Lowry procedure.

For those not familiar with the BCA protocol, it has a working range from 
10- 2000 ug/ml and yields a slightly non-linear curve, such that I 
usually construct two lines for interpolating samples, eg 10-200 ug/ml 
std. set and 200-2000 ug/ml std. set.  For those wishing only to 
construct one curve, the non-linear nature of the curve seems to be 
reduced upon extended incubation of the reaction (beyond 30 min at 37 
Celsius). 

Hope some of you find this useful,

Comments welcome,

Jeffrey W. Chisholm
Lipoprotein Research Group
Dept. of Biochemistry
Dalhousie University
Halifax, N.S., Can.

Email: JWChish at ac.dal.ca




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