Ron-- I routinely use the PCT (pre crystallization test) to see if the protein concentration is right for crystallization screening. That way you do not waste too much protein in setting up screens where everything crashes out.
What else is in your protein buffer besides Tris pH 8? You might need to add reducing agent, salt, glycerol, propanediol etc to keep it happy & in solution. It is also possible you might need to change the buffer to something else besides Tris for your crystallization trials. We sometimes use the Thermafluor assays to find additives to stabilize difficult proteins. Hope this helps! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) [EMAIL PROTECTED] "Ron hudson" <[EMAIL PROTECTED]> Sent by: "CCP4 bulletin board" <[email protected]> 20-Sep-2008 10:59 Please respond to "Ron hudson" <[EMAIL PROTECTED]> To [email protected] cc 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
