AMINO ACID ASSAY

Phil Robinson mdpjr at cc.newcastle.edu.au
Tue Jun 18 15:18:37 EST 1996


I found the following somewhere, can't remember where.  It was
designed to check if you have successfully reduced your oxidised Cys.
It seems to be a "standard" method.  I haven't tried it yet, but hope
it helps...

A quick assay to measure free sulfhydryls in peptides before and after
reduction of the peptide.
- Prepare a solution of 4 mg DTNB (Ellman's reagent,
5,5'-dithiobis(2-nitrobenzoic acid)) in 1 ml phosphate buffer, 0.1 M
Na2HPO4, pH 8.0.
- Mix 2 ml phosphate buffer, 0.1 ml peptide solution (at 0.5 - 1
mg/ml), and 0.1 ml DTNB reagent.
- Incubate 15 min, read absorbance at 412 nm.  (Make sure solutions
with NaBH4 have been acidified as this gives a false colour).  The
reaction is near stoichiometric.
Phil			Newcastle, Australia

>I am looking for a wet chemistry assay for sulfhydryl compounds,
>particularly for the amino acid cysteine and homocysteine.  Would anyone
>know of a web site for chemical analyses or of reference for the assay
>of sulfhydryls.





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