| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | | |
|
|
Re: [ccp4bb] Solubility of ligands - Summary |
|
CCP4bb navigationCCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007Subject: Re: Solubility of ligands - Summary From: "Ibrahim M {- dot -} Moustafa" I {- dot -} moustafa {- at -} PSU {- dot -} EDU Date: 2007-03-05 Two days ago I posted the question given below regarding the solubility of ligands for crystallization and activity assays. Thanks to everybody who replied to my question. I'm giving the summary of the answers below: I like the Anthony's approach, I think I'll follow it. 1) Madhevi It looks like you do not have problems in terms of crystallizing the compounds with your protein. I do not know what assay you are going to do. In our case, the compounds dissolve in DMSO. So we can get the actual concentration of the solution. In one of the assays, we do not have any problem. But in other assay (ITC), the moment we add the compound solution into the ITC buffer, the solution turns white/precipitate. In that case we are unable to do the assay. The solvents you use depend on the kind of assay too I guess. 2) Anthony First suggestion: try to work with the least amount of compound, say 0.5-1 mg in 5-10 ul for the first few solvents. But if you still want to use all your sample follow this procedure to undo the damage. If your compound is not so soluble in DMSO and probably in water and if you want to extract your compound, my suggestion is just dump 10 or more equivalents of water into the tube which results in complete precipitation of your compound. Then spin down the sample and remove the supernatant. You may have to wash few more times with water and vacuum dry your sample. It is a technique commonly used in organic chemistry labs. 3) Steve, Evaporation is a reasonable approach to remove DMSO, but you may be left with residual. Aside from HPLC purifying the compound, I can't think of any other way. You may wish to try DMF (dimethyl formamide) for those with poor solubility. Methanol in extreme cases, as proteins don't generally like it. 4) Juergen (Thanks for the paper) Consider PEG400 as your solvent too, since many crystals are grown anyhow in PEG conditions it's easier to add to the crystal then. We have success with DMSO, but sometiimes we change to PEG400 too (unpublished yet). Here's a paper which might have some useful information for you. thanks for the useful suggestions Ibrahim >At 11:28 AM 2/28/2007, you wrote: >>Dear all, >> >> I have a small library of In-silico screened compounds to test >> for activity and for crystallization trials with our protein of interest. >> >> We only have about 10 mg/ml of each compound. As there is no >> available experimental information about solubility of these >> compounds, I have no choice but to try different solvents. >> >> The first solvent to try will be DMSO (100%) to make the highest >> stock concentration of each compound. My question to those who >> passed through similar experience is: >> >> Assuming some of the compounds turned to be insoluble in DMSO, >> which is possible, how to completely recover the compounds from >> DMSO before trying another solvent. >> >> Will spinning and leaving the tube open over the bench be enough >> to get rid of the solvent or what people usually do in that case? >> What is the recommended solvent to try next? >> >> Is there a standard protocol to follow for the case we have?? >> >> thanks in advance for those who are willing to share their experience. >> >> regards, >> Ibrahim >> >>Ibrahim M.Moustafa, Ph.D. >>Pennsylvania State University >>Biochemistry & Molecular Biology Dept. >>201 Althouse Lab. >>University Park, PA16802 >> >>Tel (814) 863 8703 >>Fax (814) 865 7927 > >Ibrahim M.Moustafa, Ph.D. >Pennsylvania State University >Biochemistry & Molecular Biology Dept. >201 Althouse Lab. >University Park, PA16802 > >Tel (814) 863 8703 >Fax (814) 865 7927 Ibrahim M.Moustafa, Ph.D. Pennsylvania State University Biochemistry & Molecular Biology Dept. 201 Althouse Lab. University Park, PA16802 Tel (814) 863 8703 Fax (814) 865 7927 CCP4bb navigationCCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007 |
| ProteinCrystallography.org: Copyright 2006-2008 by Quid United Ltd |