How to measure total protein conc. of cultured-cell lysate?

Martin Offterdinger a8803349 at unet.univie.ac.at
Thu Dec 5 03:10:46 EST 1996


On Wed, 04 Dec 1996 12:17:37 -0500, Mohamed BaSalamah
<mbasal at med.unc.edu> wrote:

>Hi
>
>I'd appreciate all help in solving the following problem.
>
>I want to ensure equal loading of two different cell lines-cell lysates
>for western analysis. That's why I want to have total protein conc.
>
>  The media for these cells contain phenol red.
>  The lysis is done using 2% SDS.
>  The protein of interest is likely to be glycosylated.
>
>If someone who has done this type of thing would share with me his/her
>protocol, or point out critical parameters that I might've been over
>looking, I'd be grateful.
>
>Thanks
>
>Mohammed BaSalamah
>Grad. Student
>mbasal at med.unc.edu
Dear Mohammed
in my opinion it is almost impossible to make a protein assay if you
use SDS as lysis detergent-how about using some non-ionic detergent
such as NP40 , Brij , Triton X-100, ....? You could centrifuge off the
nuclei and afterwards do a Bradford assay using the Bio-Rad reagent
e.g.-but this will not work with SDS, as SDS itself reacts with the
Bradford reagent.This assay will also work for glycoproteins as the
dye used here is adsorbed to amide bonds of the protein!
Hope this helps-  Martin



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