can't you try different volumes: i.e. 360 nl protein solution in very little buffer and salt + 40 nl precipitant. Should give roughly 10 mg/ml after equilibration.
Best wishes
Kornelius
On Thu, 24 Apr 2008 12:42:42 +0100
Roberto Steiner <[log in to unmask]> wrote:
> I have a similar problem Matthew.
> Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see.
>
> Regards,
> Roberto
>
> On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote:
>
> > Dear All,
> >
> > I have a 50kDa protein that is soluble and monodisperse at up to
> > approx 1mg/ml (after Ni-affinity and size-exclusion chromatography).
> >
> > However, it aggregates (probably both via disulphides and via
> > 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/
> > ml, even in the presence of several detergents. I don't want to add
> > DTT since my protein should have several intramolecular
> > disulphides....although I do have 2 free Cysteines, partially
> > exposed. I have already tried mutating the Cysteines, with little
> > improvement.
> >
> > Any suggestions for obtaining 5-10mg/ml?
> >
> > Does anybody have good experiences with usin L-Arg and L-Glu (e.g.
> > At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS,
> > 2004, pages 8933...)
> >
> > Thanks for any input!
> >
> > Yours,
> >
> > Matt
> >
> > ====================================
> > Matthew J. Bottomley, Ph.D.
> > Senior Research Biochemist
> > IRBM / MRL-Rome
> > ====================================
> >
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> ---
> Dr. Roberto Steiner
> Randall Division of Cell and Molecular Biophysics
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----------------------------------------------
Kornelius Zeth
Max Planck Institute for Developmental Biology
Dept. Protein Evolution
Spemannstr. 35
72076 Tuebingen, Germany
[log in to unmask]
Tel -49 7071 601 323
Fax -49 7071 601 349
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