With information you've provided I have multiple suggestions for you, some of which you may have already tried:
1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER <r.grinte...@research.gla.ac.uk > wrote: > Hi All, > > A quick question if you've ever worked on membrane proteins, I'm trying to > optimize crystals for bacterial integral outer membrane protein I'm working > on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH > 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of > birefringent gel forming in the same condition, I get the feeling that this > is Detergent/Protein complex and is robbing my crystals of material to grow > bigger. > These crystals diffract to 5 A so I'd quite like to make them bigger and > better, > > Cheers, > > Rhys -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures <http://www.emeraldbiostructures.com/> Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman....@gmail.com jfair...@embios.com
