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Thiol determination by DTNB and detergents

Thiol determination by DTNB and detergents - Protocols and Methods Forum

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  #1  
Old 01-30-2004, 04:57 PM
SB
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Default Thiol determination by DTNB and detergents



Hi

Does anyone know what effect detergents (eg: SDS at 10 or 15%) have on
the measurement of thiol concentration using DTNB?

Thanks

SB
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  #2  
Old 02-01-2004, 08:06 PM
Sell Biology
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Default Thiol determination by DTNB and detergents

"SB" <[Only registered users see links. ]> wrote:


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.
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  #3  
Old 02-02-2004, 03:08 PM
SB
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Default Thiol determination by DTNB and detergents

Sell Biology wrote:


Thanks for your suggestion. Actually I was thinking of whether it will
be possible to solubilize cells with detergent and then use DTNB to
estimate thiol content. All studies on thiol determination use acid
precipitation of proteins and estimating thiols in the acid soluble
supernatant. I welcome any input regarding this matter.

SB
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  #4  
Old 02-02-2004, 11:25 PM
D.K.
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Default Thiol determination by DTNB and detergents

On 2 Feb 2004 15:08:15 GMT, "SB" <[Only registered users see links. ]> wrote:


SDS does not interfere with DTNB but unfolding of proteins
under non-reducing conditions favors S-S bons formation. We
see this artefact in running "non-reducing" gels all the time.

If you are after low MW thiols, you really have to do acid
precipitation. If, OTOH, you want to estimate total free
accesible thiols, lysing cells with non-ionic detergent like
Triton X-100 and then doing DTNB reaction makes more sense.

DK

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  #5  
Old 02-04-2004, 12:01 PM
Dr Engelbert Buxbaum
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Default Thiol determination by DTNB and detergents

Sell Biology wrote:



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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detergents , determination , dtnb , thiol


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