unwanted ppt in Biorad assay

larson eric elarson at ux1.cso.uiuc.edu
Sat Sep 10 01:19:23 EST 1994


hm10006 at mole.bio.cam.ac.uk (Hemlata Mistry) writes:

>Help!

>I get a large precipitate in a Biorad protein assay.  Maybe my assay
>conditions are not right. I have been extracting cAMP from Drosophila eggs
>using 6% TCA or hot 0.1M HCl, and saving the pellets containing protein +
>eggshell. I resuspend the pellet in deoxycholate, spin down undissolved
>stuff and try to assay amount of protein in supernatant. All I have got so
>far are massive blue pptes, whose OD I cannot measure! Any suggestions?
=============
 
The DOC is the problem.  DOC is a wonderful way to ppt. proteins since it
will fall out of solution in the presence of acid.  The Biorad reagent is
in phosphoric acid, thus the source of your problem.  Similarly, the Biorad
reagent will react with many detergents giving a blue color.
 
I recommend just doing a Lowry assay with the ppt. protein (or a Larson
Lowry :-). 
 
You can try resuspending the pellet with a small amount of NaOH and then do
a Biorad assay if your protein concentrations are very low.  Skip the DOC
if you do this.
 
-- 
Eric Larson                  | University of Illinois at Urbana-Champaign
USDA/Agronomy                | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu     | Voice 217.244.3079  Fax 217.244.4419
Fidonet: 1:233/4.1           | My opinions are my own, but correct :-) 



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