Thanks for all the suggestions. I forgot to mention that I had tried microseeding and it was partially successful. I am trying this while also pursuing other venues. At Florian's suggestion I have set up a free interface diffusion method and it looks very promising. I have a few free floating crystals that seem somewhat flawed, but otherwise very nice. I will try combining microseeding techniques and free interface diffusion to make THE crystal.
Timur On Mon, Jul 18, 2011 at 11:01 AM, Florian Sauer <[email protected]>wrote: > Dear Timur, > > one possibility to handle this problem can be the change from vapor (I > assume this is what you do) to the free interface diffusion method. Phase > separation often occurs if the protein is immediately exposed to the full > precipitant concentration while it might not "escape" into its own phase if > it gets slowly equilibrated. > There are commercial setups available for this method but you can also do > it in a normal vapor diffusion plate. > To do so, just put the protein and precipitant drops next to each other and > then link them through a thin liquid bridge. Requires some practice and > works best with large drops but helped me in several similar cases. > > > Good luck! > > Florian > > > > Am 18.07.11 15:52, schrieb F. Timur Senguen: > > Hi everyone, > > I have been issues with a particular protein. I have been close for a > while, but yet so far. > > Rather than going from a clear drop to crystal, my protein first > undergoes phase separation (large oily drops) in which one phase contains > most, if not all, of the protein. This phase separation occurs within a day > of preparing the drop. A day after phase separation the oily phase is now a > large disordered crystalline mass which does not diffract very well. I have > tried changing buffer concentrations, precipitant amounts, ionic strengths > and pH and in all cases this phenomenon is observed. I even screened protein > concentrations to see if reducing protein concentration would prevent the > phase separation. > > Is there any way to bypass this phase separation, which I think prevents > me from obtaining nice crystals. Should I try detergents, chaotropes, or > other additives? > > Thanks in advance. > > Timur > > -- > F. Timur Senguen, Ph.D. > Postdoctoral Research Fellow > Boston Biomedical Research Institute > 64 Grove Street, > Watertown, > MA 02472 USA > > > -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA
