You could assay some of the suggestions involving small molecules etc. quickly by using the Tm shift assay, if you had the equipment handy?
Analytical Biochemistry Volume 357, Issue 2, 15 October 2006, Pages 289-298 Thermofluor-based high-throughput stability optimization of proteins for structural studies Ulrika B. Ericssona, B. Martin Hallberga, b, George T. DeTittac, Niek Dekkerd and Pär Nordlunda, Does your enzyme have any known cofactors, metals etc. which you know bind? Stick some of them in during purification to see if they can stabilise your protein. A more simple suggestion, I tried this recently with some success, essentially sticking 0.2M Arg / 0.2M Glu in the buffer. I had no glycerol in there. J Am Chem Soc. 2004 Jul 28;126(29):8933-9. A simple method for improving protein solubility and long-term stability. Golovanov AP, Hautbergue GM, Wilson SA, Lian LY. Just as a related aside..... the MCSG (I think) just presented a poster at the Keystone symposia on structural genomics where they systematically tested a selection of their targets, showing how glycerol was absolutely required in some cases, but in other cases absolutely guaranteed failure, with yet other examples not being bothered whether it was present or not. The reaction from some to this poster was interesting with one grad student in particular remarking "My PI would kill me if she saw me making a buffer without glycerol!". cheers, charlie On Tue, Feb 23, 2010 at 3:07 AM, SERAH KIMANI <serah.kim...@uct.ac.za>wrote: > Dear all, > > Does anyone have an idea of something else that I can use instead glycerol > to maintain solubility of my protein? I have been having 10% glycerol in my > protein solution and this has helped me get crystals in like three different > conditions. However, I would want to get crystals of mutants-substrate > complexes, but unfortunately glycerol interferes with the binding of the > substrates to my protein. So, for the complexes to form, glycerol has to be > out (I have tested this using the wildtype enzyme both in the presence and > absence of glycerol). Now, in the absence of glycerol, I get a lot of > precipitation, and no crystals even after optimizing around the known > conditions with different protein concentrations. I have tried re-screening > for new conditions in the absence of glycerol, but I haven't found any > condition that yields crystals in the absence of glycerol. > > I was wondering if anyone might know of something else that I could use in > place of glycerol in my protein solution?? > > Regards, > > Serah > University of Cape Town > > ______________________________________________________________________________________________ > > > UNIVERSITY OF CAPE TOWN > > This e-mail is subject to the UCT ICT policies and e-mail disclaimer > published on our website at > http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from > +27 21 650 4500. This e-mail is intended only for the person(s) to whom it > is addressed. If the e-mail has reached you in error, please notify the > author. If you are not the intended recipient of the e-mail you may not use, > disclose, copy, redirect or print the content. If this e-mail is not related > to the business of UCT it is sent by the sender in the sender's individual > capacity. > > _____________________________________________________________________________________________________ > >
