Hi Venkat, I think there are maybe a few things you could do.
- Have you tried to lower the concentration of your protein? Maybe these clusters appear like so because there is little time for better ordered growth. If you decrease the protein concentration, supersaturation will occur slower and your crystals will have a chance to grow slower too and you will also have less nucleation... Also, for checking that the concentration is the right one there is a Pre-crystallization test available from Hampton, with 4 reagents I think, that will give you clues regarding which concentration to use. - Seeding? If you could crush these things and then use them as seeds in drops in which the precipitant concentration is a bit lower than the usual one in which you get the crystals, you will introduce nuclei that will only grow...maybe not super beautiful after the first seeding, but after a few ones, for sure! It is also helpful to try different dilutions of the seeds, so that you don't introduce too many or too less nuclei in the drop. - Diffusion rate control by placing a layer of oil between the reservoir and your drop (or you can also place the oil directly on the top of your drop). This will also lead to supersaturation but slower... you can play here with the type of oil (Silicon, paraffin or Al´s) and also with the thickness of the layer of oil. Thicker layer of oil will allow slower equilibration. - Temperature... at which temperature do you have your crystals? In my case I have always got better ones at lower temperatures than at room temperature. Maybe this could also help. Hope this helps! Good luck and best wishes, Almu 2014-05-21 11:58 GMT+02:00 venkatareddy dadireddy <[email protected]>: > Dear All, > > I'm working on 24.3 KDa protein (pI 4.84) with (Asp + Glu): 36 and (Arg + > Lys): 16. Protein is in buffer Tris (10mM), pH 7.5. I got sea urchin like > crystal clusters @ protein conc 38.5 mg/ml in sitting drop method; > condition 0.2M NH4I, 20% PEG3350 & pH 6.8 (PEG/ION A12). These crystals are > very thin. I tried different pH values ranging from 6.0 -9.0 with 0.5 > units increment, PEG conc. from 10 -30% and NH4I conc. from 0.1 -0.5 M > independently in hanging drop method and ended up with NO Crystals. Can > somebody suggest strategy to improve crystals. Please find the crystal pic > with attachment. > > Regards, > Venkat. > -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
