RE: [ccp4bb]: Skin formation in crystallization trials:

[Patrick Shaw Stewart]

Muthu   One obvious thing to try is to crystallize under oil.  Either put a few microlitres of oil on top of regular sitting drops, or use microbatch-under-oil (see e.g. http://www.douglas.co.uk/microbatch.htm )   Since you want to minimize skins I would cover the drops with pure paraffin.  If you need to increase the rate of equilibration (or evaporation) use up to 50:50 silicone and paraffin (Hampton’s Al’s Oils).  Microbatch is sometimes helps a lot in reducing skins on the surface of drops, e.g. Pearl et al. EMBO Journal 13. (1994), p 5810.  Surprisingly, the oil also seems to reduce oxidation of protein within the drop too. Patrick --  [EMAIL PROTECTED]    Douglas Instruments Ltd.  DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090    US toll-free 1-877-225-2034 From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: 22 June 2006 21:37 To: ccp4bb@dl.ac.uk Subject: [ccp4bb]: Skin formation in crystallization trials:   I am working on a protein with three domains. I have purified three different constructs – only one domain or two domains or all the three domains. All these proteins, I expressed as MBP fusion and purified well and runs as monomer in gel-filtration. Surprising all these proteins form skin in almost all conditions (PEG or salt) of Nextal and Hampton crystallization screen. These domains have some cysteines,and ddition of reducing agent (DTT or TCEP) during crystallization trials did not help and I still get skins. The proteins run as monomer in gel-filtration column even with out any reducing agents. Searching in Web did not give me clear answer. Any body has any such experience/suggestions.   Thanks muthu