SDS and Protein Quantification

Jonathan B. Marder MARDER at agri.huji.ac.il
Fri Jul 23 04:09:17 EST 1993


In article <Mills_j-230793170950 at pharm11.med.upenn.edu> Mills_j at a1.mscf.upenn.edu (Jason Mills) writes:
>Subject: SDS and Protein Quantification
>From: Mills_j at a1.mscf.upenn.edu (Jason Mills)
>Date: 22 Jul 93 22:00:17 GMT
>
>     Just wanted to ask if anyone out there had a good method for
>determining the amount of protein in cell extracts containing 2% SDS.  I've
>been researching the literature up and down and have only been able to come
>up with two solutions: 1) do Lowries (which we aren't set up to do) 2) do
>some sort of TCA precipitation and dot blotting (which we were set up to do
>until our dot-blotter got broken).  I only have one set of samples with
>this level of SDS and, thus, am trying to avoid anything I would have to
>buy more reagents for.
I routinely do dot protein assays on blotting paper with the sample in 2-3%
SDS buffer ready for electrophoresis.  The sensitivity to low protein
concentration is poor (minimum ~0.1 mg/ml) but I can go as low as a half
microlitre per dot.  I list below references for the original dot-blot idea
(1) plus my own modifications (2) which deals specifically with SDS-
dissolved samples.  You can adapt the method pretty freely - e.g. do the
staining using with isopropanol-dissolved Coomassie if that's what you have
ready for staining gels.

1. Esen, A. (1978) Analytical Biochem. 89, 264
2. Marder, J.B., Mattoo, A.K. and Edelman, M. (1984) Methods in Enzymol. Vol
   118, pp. 384-396 (blot method on p. 390)

>     Interestingly, I thought I found the answer a couple of days ago when
>I saw a paper describing a modified Bradford which was supposed to --
>according to the paper which referenced it -- be able to handle large SDS
>concentrations.  Only after I ran to the library with bated breath and read
>the paper 5 times over did it sink in that they were saying the exact
>opposite -- that they couldn't get any modification of the Bradford to work
>in the presenced of SDS.  You just can't trust what youe read anymore...
>
>Jason
>
>Mills_J at a1.mscf.upenn.edu
__
Jonathan B. Marder                 '
Department of Agricultural Botany  |     Internet: MARDER at AGRI.HUJI.AC.IL
The Hebrew University of Jerusalem | /\/ Bitnet:   MARDER at HUJIAGRI
Faculty of Agriculture             |/  \ Phone:    (08 or +9728) 481918



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