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

Bernard Murray bernard at elsie.nci.nih.gov
Thu Dec 5 13:19:10 EST 1996


In article <32a68241.2633983 at news.univie.ac.at>, a8803349 at unet.univie.ac.at 
says...
>
>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.
>>Mohammed BaSalamah

>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

I believe that it is possible to assay protein in the presence of SDS
as long as the protein standards for the calibration curve contain
the SDS (and any other interfering agents) at the same final
concentration.  For some assays (especially involving dye binding) the
presence of the SDS "sensitises" the assay and increases the slope of
the line (don't some of the Bradford protocols *include* SDS in the
reagent?).  The booklets with the BCA protein assay kit (Pierce) and
the Lowry protein assay (BioRad) claim that they are insensitive to SDS
as high as 1% so these may be good choices if you don't want to make up
"special" standards.  The only thing to be wary of is cloudiness in the
detergent-containing samples - I have a vague recollection that this can
be reduced by adding a small volume of chloroform (??).  The phenol red
may actually be more of a problem so it would be worth changing the
culture medium to the equivalent without this if you can.
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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