Hi everybody,
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(unpublished yet).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
