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

David Winterbourne sghk100 at sghms.ac.uk
Fri Dec 6 04:27:30 EST 1996


In article <5873mu$haq at light.nih.gov>, Bernard Murray says...
>>in my opinion it is almost impossible to make a protein assay if you
>>use SDS as lysis detergent
...
> 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.



It is possible to get good protein quantitation in most buffers (including SDS
buffers with mercaptoethanol and bromophenol blue). I developed an assay which precipitates the 
protein on filter paper, leaving most interfering substances (detergents, salts, dyes etc.) in 
solution. The Sensitivity is 0.5-8ug and can be applied to cell extracts in SDS sample buffer.

Stain 1-8ul spots placed on a grid of 1cm squares on Whatman 3MM for 1h with
0.04% Coomassie blue R250 in 25% ethanol, 12% acetic acid.
Include blanks and standards on the same sheet.
Destain with 10% ethanol, 5% acetic acid.
After drying the sheet, cut the squares out, place in tubes and elute with 1ml
1M potassium acetate in 70% ethanol for 1h.
Measure OD 590nm.

For full details and a general discussion, see:

Winterbourne, D.J. Chemical assays for proteins. In: Methods in Molecular
Biology. Volume 19. Biomembrane Protocols I. Isolation and Analysis, edited by
Graham, J.M. and Higgins, J.A.Totowa, New Jersey:Humana Press, 1993,p.
197-202.


-- 
Dr. David Winterbourne
Department of Surgery
St George's Hospital Medical School, London SW17 0RE, England
Tel: 0181-725-5581   Fax: 0181-725-3594




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