One method that worked for me was to dissolve my ligand in 100% DMSO, as
suggested in the previous response, then add a 3 molar excess of ligand
to protein so that the final concentration of DMSO in the protein-ligand
solution was no greater than 10% - of course the maximum concentration
of DMSO that your protein can suffer will be  protein specific but you
could investigate this by incrementally adding DMSO to just a solution
of your protein at your working crystallisation concentration then
measuring scattering at 600nm in a spectrophotometer to determine the
critical DMSO concentration that causes your protein to precipitate (if
you have sufficient protein to 'waste'!). If your ligand binds tightly
to your protein at an equimolar concentrations you can then remove
excess ligand and DMSO by passing your sample through a G25 Sephadex
column. Hope that helps.

Rob Hussey.

>>> David Briggs <[log in to unmask]> 12/12/07 7:19 AM >>>
The review cited below is a great source of ideas to help you get your
ligand in your crystals

Hassell, A et al
Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):72-9.
Crystallization of protein-ligand complexes.

Hopefully it may help you!

Dave

On 11/12/2007, Schubert, Carsten [PRDUS] <[log in to unmask]> wrote:
> Simon,
>
> solubilize your ligand in DMSO so it is maximally concentrated, 100mM
works fine. Add enough compound to achieve 2-3 fold excess to your
protein, mix and set up. Make sure your final DMSO concentration is ~3%,
otherwise chances are you might harm your protein. If you cannot achieve
a high enough stock concentration of DMSO to be below the 3% threshold,
dilute you protein in the storage buffer to ~1mg/ml. Add compound to 2-3
fold access, incubate and co-concentrate to the desired concentration.
That way you avoid the DMSO shock.
>
> Alternatively you could incubate the concentrated protein with the
compound solubilized in water for 24-48hr and hope it is soluble and
potent enough to get taken up by the protein and then set up your trays.
>
> HTH
>
>         Carsten
>
>
>
> > -----Original Message-----
> > From: CCP4 bulletin board [mailto:[log in to unmask]]On
> > Behalf Of Yue Li
> > Sent: Tuesday, December 11, 2007 11:55 AM
> > To: [log in to unmask]
> > Subject: [ccp4bb] insoluble ligand
> >
> >
> > Hi all,
> >
> > I have one ligand which is insoluble in water, and I would like to
> > co-crystallize it with my protein. Is there any other method
> > except for
> > dissolving it in DMSO ?
> >
> > Thanks
> >
> > Simon
> >
> >
>



-- 
============================
David C. Briggs PhD
Father & Crystallographer
http://personalpages.manchester.ac.uk/staff/David.C.Briggs/
AIM ID: dbassophile
============================


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