SDS quantitation and removal with resins

Frank R. Gorga fgorga at bridgew.edu
Tue Jul 8 06:45:04 EST 2003


Could the Pierce reagent simply be a solution of KCl or KAc?

Potassium dodecyl sulfate is fairly insoluble in water. Thus one can remove
most of the "SDS" from a solution simply by adding  ~0.75 M K+ to a sample.
I do not have a clue what the final concentration of detergent will be... it
will vary greatly with other components present. But it might work in your
application.

As for using various resins... all have the potential problem of absorbtion
of the protein of interest to the resin. This is not usually an
insurmonttable problem, just beware. (I have had good luck in reducing loss
of protein when removing non-ionic detergents using "Biobeads" by adding
"extra" lipid to the mix. Not an ideal fix, but one does what one has to!)

As for quantitation of detergents... not easy! The fluorescence assays,
while they work well to measure CMC in a simply solution are not specific
enough for a sample containing biological molecules.

In the "old days" (late 70's), one could by radiolabeled SDS and TX-100 from
NEN. (I don't know if they are still available.) This was the simplest way
to follow the removal of detergent... add a 1000 cpm / uL of 3H-detergent to
the starting sample and count small samples along the way.

--- FRG

Frank R. Gorga, Dept. of Chemical Sciences, Bridgewater State College


"Scott Coutts" <scott.coutts at med.monash.edu.au> wrote in message
news:3F09F59D.70108 at med.monash.edu.au...
>
>
> Dr Engelbert Buxbaum wrote:
> > Scott Coutts wrote:
> >
> >
> >>Hi!
> >>
> >>Does anyone know of a way that I can assay for SDS concentration in a
> >>sample containing protein? I want to know how much detergent remains
> >>after attempts at removal. I need to assay for regular (non-labelled)
> >>SDS, by the way.
> >>
> >>Also, I'm thinking of removing the SDS from some protein samples using a
> >>resin, perhaps Dowex 1-X2 or 1-X8 or similiar.
> >
> >
> > You can assay for any detergent above cmc by measuring the fluorescence
> > of DPH (1,6-diphenyl-1,3,5-hexatriene), which is non-fluorescent in
> > water but highly fluorescent when dissolved in micells (358/450 nm).
> > This is also a nice way to measure the cmc (Chattopadhyay & London,
> > Anal. Biochem. 139 (1984) 408-412).
> >
> > For the removal of detergent from biological samples you can use
> > hydrophobic beads like BioBeads SM-2 (Philippot et al., BBA 734 (1983)
> > 137-143), there are also various recent papers by the group of Rigaud
> > which are worth reading, for example Eur. Biophys. J. 27 (1998)
> > 305-319). Pierce has a similar product, packed in little disposable
> > columns.
> >
>
> Hi,
>
> THanks for the info. I think the >CMC fluorescence method is as close as
> I'm going to get to an assay method... unfortunately, I dont think it's
> going to be usefull for me.
>
> I agree that the hydrophobic bead method may be the best bet for
> removal. I knew about the pierce product already (if you mean the
> SDS-Out product), and had wondered what they use in that. They say it's
> a 'precipiation reagent'... but the spec sheet and instruction 'manual'
> on their website doesnt really give any information about whats in it or
> how it works. Have you used it before? You say that it's column-based;
> does it use hydrophobic beads in that or do they rely on a (at leatst
> semi-) specific precipitation of SDS?
>
> Anyhow, thanks for the info.
>





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