What protein concentration are you using in your screen? It sounds like you need to try lower protein concentration, or a screen with lower precipitant concentration, or both. Hampton makes a Crystal Screen Lite with the precipitant concentrations half of what they are in the normal screen (or you can dilute the regular screen). Don't forget that protein concentration is one of the important variables to optimize. For some complexes, I have had best results with protein concentration as low as 2.5-3 mg/mL. Evette
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) 953-0046 (lab) ________________________________ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ron hudson Sent: Saturday, September 20, 2008 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein complex crystallisation Dear CCP4 Community, My apologies for the off topic question to the bb. I am trying to crystallise a small protein-protein complex. I purify as a complex protein after expression in BL21DE3 cells through NI-NTA affinity chromatography. pI of one of the protein is around 10.6 where as another component have 4.0. I am in the screening stage of the crystallisation. During crystallistion process it precipitate very quickly as i add the protein into the crytallisation solution drop. This happens in almost all the condition of the hampton INDEX and crystal screens. I purify this complex in Tris-Cl buffer at pH=8.0. it is happily soluble and donot prcipitate at this pH in the same buffer. I can concetrate this protein upto ~10-15mg/ml. could any one suggest the solution, that will be most appreciatable. Thanks in advance Ron
