Protein quantification with Ammoniumsulfate present?!

David Winterbourne sghk100 at sghms.ac.uk
Thu Jun 4 03:58:52 EST 1998


Some time ago, I developed a Coomassie blue protein assay. Although I
haven't tested it directly with ammonium sulphate, I would expect the
assay to work perfectly well. The principal is to spot aliquots onto a
grid of squares drawn on Whatman 3MM. The sheet is then stained with
Coomassie blue, destained and quantitated by eluting the bound dye, giving
sensitivities down to ~60ug/ml. Although the sensitivity is not quite down
to the level you require, it is often possible to detect spots of lower
concentrations in a semiquantitative way just by looking at the stained
and destained sheet (i.e. without eluting the dye).

The assay is unaffected by 1M HEPES, 1M tris, 2M sucrose, 1% SDS and 9M
Urea (although fixing the protein in 10% TCA before staining may be
required to completely remove minor signals from the most strongly
interfering substances).

The details are in:

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.

and another description of the reagents and general outline (but mostly as
applied to cells in microtest plates) in:

Winterbourne, D.J. Cell growth determined by a dye-binding protein assay.
Biochem.Soc.Trans. 14:1179-1179, 1986.

-- 
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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