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 <[EMAIL PROTECTED]> 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] <[EMAIL PROTECTED]> 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:[EMAIL PROTECTED] > > Behalf Of Yue Li > > Sent: Tuesday, December 11, 2007 11:55 AM > > To: CCP4BB@JISCMAIL.AC.UK > > 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 ============================ The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
