Hi everyone, Thank you for your responses I will keep on with optimisation!
Cameron -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CAMERON FYFE Sent: 11 December 2013 14:57 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystallisation optimisation query Hi everyone, I have been working with a protein 182 kDa in size and the protein has been purified in 200 mM NaCl, 50 mM Tris, and pH7.5. After incubating with protease and purifying by size exclusion I have taken one of the main peaks and managed to get good looking crystals with the largest being 1 mm long and 0.2 mm wide with variable thickness. When the SEC purified peak is run on an SDS PAGE gel there are multiple bands with a combined molecular weight that adds up to the original proteins size and does not contain the protease used (verified by MS). The successful crystallisation condition contains 100 mM Potassium Chloride, 100 mM HEPES pH 7.5, 25% SOKALAN CP 7. We are using a cryo condition using 30% Glycerol (anything lower has water rings) and we found the crystallisation condition crashes with PEG400 and Ethylene Glycol. The initial screening condition crystal was taken to Diamond and we got diffraction to 4.9 Å. I have tested several of these crystals some of which show weak diffraction however most of which do not diffract at home. I feel the result of the protease digestion has resulted in a complex of domains held by non-covalent interactions has anyone any advice for optimising crystals like this? SOKALAN CP 7 seems to be a relatively exotic precipitant with little information available online would anyone have any experience of using it or even some information on its structure? Thanks, Cameron Cameron Fyfe Institute of Infection, Immunity and Inflammation Sir Graeme Davies Building 120 University Place University of Glasgow Glasgow G12 8TA Tel +44 (0)141 330 6481 c.fyf...@research.gla.ac.uk
