Re: [ccp4bb] Multiple nucleation
In addition to trying lower concentrations of PEG and protein, you might consider whether aggregation in your protein stock or vibration in your crystallization environment are contributing to the problem, and how they might be minimized. It might be obvious, but we have found that it is important for protein stocks to be filtered or centrifuged to remove aggregates immediately prior to setting up drops, and if the protein solutions have been sitting around for a few hours or a few days, they must be centrifuged again. We also had a stupid incident recently where conditions that had previously generated high quality crystals were suddenly producing thousands of tiny crystals instead, and the problem was traced to a clinical centrifuge that had been moved to an adjoining bench and was vibrating the crystallization shelf. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-2857 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jobichen Chacko Sent: Thursday, March 29, 2007 7:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Multiple nucleation Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi
Re: [ccp4bb] Multiple nucleation
I've found that crystallization in sitting drops under oil dramatically reduces the no. of nucleation events and increases the overall crystal size. Now, if the increased crystal size helps improve diffraction is something to be tested. Among the many other suggestions... Raji -Included Message-- Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi -End of Included Message--
Re: [ccp4bb] Multiple nucleation
What often works for this scenario: Change temperature. Try 21, 15, and 4C. Add glycerol or ethylene glycol - set a screen with your condition as basis, and % EG/glycerol across - from 1% to 15% in suitable increments Find additives that produce no crystals, set up tray(s) with those, then seed with your microcrystals (worked really well for me in a few cases) Reduce protein concentration Add gelling agents to the set-up Regards, Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jobichen Chacko Sent: Thursday, March 29, 2007 7:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Multiple nucleation Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi
