gel filtration chrom.: Tween 20 problems
"jphilo*NO SPAM12*" at earthlink.net
Mon Sep 14 10:34:55 EST 1998
With regard to detergent properties, there is a nice web page by Shaun
Shaun doesn't list a micelle size for Tween 20, but that for Tween 80 is
Ian MacFarlane's suggestion is an excellent one. It is also possible
that your extra peak is some sort of specific detergent-protein complex.
Some proteins do bind stoichiometric quantitities of certain detergents.
Surprisingly, other proteins for which detergents are known to work well
for limiting aggregation or surface denaturation do not seem to bind any
at all. Ted Randolph in Engineering at U. Colorado, Boulder has done
some nice studies of this using spin-labelled detergents.
John Philo, Alliance Protein Laboratories
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Ian McFarlane wrote:
> What I am suggesting is that if you think that your extra peak is Tween 20
> micelles simply inject a sample of the buffer that your biopolymer is
> dissolved in. This will show you whether the peak is associated with your
> sample or with your buffer. As a final cross check you could make up a
> fresh solution with only Tween and water to check that it is micelles and
> not another contaminant. One other thing you might investigate is that
> somewhere buried in the literature will a be an average value for the
> number of Tween-20 molecules per micelle which should roughly correspond
> to the M.Wt. of the contaminant peak, but I can't help you with where you
> might find this.
> Perhaps somebody else on the newsgroup could help?
> Ian Mc
> In article <6t8mc6$s0c$1 at nnrp1.dejanews.com>, dan.dille at hmrag.com wrote:
> > Ian, I appreciate your response. Could you please be more specific about
> > injecting buffer?
> > In article <i.mcfarlane-0909981215070001 at 126.96.36.199>,
> > i.mcfarlane at icrf.icnet.uk (Ian McFarlane) wrote:
> > > Dan try injecting an aliqout of the buffer and you will have your answer.
> > >
> > > In article <6t3rdl$p6$1 at nnrp1.dejanews.com>, dan.dille at hmrag.com wrote:
> > >
> > > > Currently use GFC to monitor a biopolymer product. The product formulation
> > > > contains Tween 20 above the CMC. This results in a small chromatographic
> > peak
> > > > appearing between the biopolymer's dimer and monomer peaks. It interfers
> > with
> > > > quantitation. Since it elutes between the column's void and
> exclusion volume
> > > > and only occurs when the sample contains Tween 20, I'm assuming that
> its the
> > > > micelles of Tween 20. Does anyone have a similar experience and possible
> > > > solution? Conditions:50mM Na-phosphate, pH6.8, 100mM NaCl Mobile Phase,
> > > > Tosohass G3000SWxl column, UV210nm detection, 200mcL injection volume
> > > > Dan
> > > >
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> > >
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