Dear Jobi, See if you can slowly increase the PEG concentration to 28 - 30% and that will be a good cryo. Since BIS Tris Propane is the buffer, I think, 28% 3350 should work fine.
If the crystals crack by going straight to 28% PEG 3350, 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate solution, put the crystal in a 24% solution and slowly increase the concentration by adding the higher concentration well solution. It seems to be a good idea to keep the crystals for 2 or 3 mts in a solution before increasing the PEG concentration (well, this seems to vary with protein). Also remember to include any salts in the cryo (at least half of the concentration) that may be already in the protein solution. Kind regards, Mathews -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Jobichen Chacko Sent: Wednesday, April 13, 2011 2:44 AM To: [email protected] Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
