Hi One of the easiest things to try is in-situ proteolysis - chymotrypsin (1:1000 or so, we add 1ul of a 1mg/ml solution of chymotrypsin in H20 to about 100ul of protein solution) is the classic - mix the protease with the protein and then set up as normal. Of course you can try other proteases as well. I would generally not recommend subtilisin or proteinase K - these are almost guarenteed to turn your protein into amino acid soup.
Matrix seeding (Allan D'Arcy is the lead author on the reference for this) with the crystals you have is another relatively painless thing to try (ie, use your crystals to seed into a screening experiment) - given that your condition is ammonium sulfate based, be aware that you will get lots of lovely crystals in any screening condition containing calcium. may the force be with you Janet ________________________________________ From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Narayanan Ramasubbu [ramas...@umdnj.edu] Sent: 10 September 2009 01:36 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help with improving these crystals Dear All: We have been struggling to improve the crystals shown in the attachment. These crystals form from a protein solution 8 mg/mL (before drop), drop sizes are 2 microL of protein + 2 microL of well solution (0.1 MES buffer, pH 6.5, 1.8 M Ammonium sulfate with 12 mM CoCl2). The protein was dissolved initially in 20 mM Tris.HCl, pH 8.0 containing 1 mM EDTA. Low cobalt also gives smaller crystals. These diffract up to 8 A. Any suggestions to improve these crystals would be most welcome. Subbu PS: We have used many additives (Hampton Research - including detergent additives) to alter the morphology but with little success.
