Thiol determination by DTNB and detergents
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Wed Feb 4 07:01:30 EST 2004
Sell Biology wrote:
> "SB" <biswas.16 at osu.edu> wrote:
> > Hi
> > Does anyone know what effect detergents (eg: SDS at 10 or 15%) have on
> > the measurement of thiol concentration using DTNB?
> I would guess none after checking PubMed with the search "DTNB AND SDS AND
> interference" and coming up with 0 items.
> But if it's really a worry for you, there are ways of precipitating excess
> SDS in solution merely by adding potassium salts (K-SDS is insoluble, as I
> recall), mixing, probably at cold temperatures, spinning it out and getting
> the supernatant.
> Assay a sample of your thiol-containing material (protein?) with the excess
> SDS present and a sample in which you've tried to remove the excess, and
> compare the results (do replicates at two or three levels for getting a
> good assessment of potential negative interferences). Your question on
> interference will then be answered.
Potassium precipitation of SDS will also remove some of the protein, the
amount will be uncontrolable.
SDS should not interfere with DTNB, but nevertheless include a reagent
blank (i.e. a sample which contains all the other constituents of your
sample except the protein). Also include positive controls (known amount
of protein with known number of thiols) in the same buffer. Then any
ill-effect of any of your sample components will be readily apparent.
If all else fails, consider precipitating your protein with
chloroform/methanol or acetone.
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