Hi Rhys, The bOG concentration of 1.0% is quite acceptable and if your protein is stable and happy in OG, then you're in luck. At 30% PEG 600, I'm not surprised you're seeing the phase separation or detergent 'blob's for lack of a better term. I believe you have the latter, which many of us working with memb proteins experience.
You are probably at above 1% OG though. When you concentrate, do you use 100kDa cutoff membrane. Based on the size of your protein, this would be a safe concentrator to use. FYI, I have several proteins that are ~50kDa, and I can use 100kDa concentrators without ill effects. If you are using 100kDa membrane, then you might try 'concentrating' via an ion-exchange column before actual concentration. Lastly, as Jim mentioned, you might want to change your construct by a few residues. This might help with the resolution. -john On Thu, May 9, 2013 at 3:49 PM, RHYS GRINTER <r.grinte...@research.gla.ac.uk > wrote: > Thanks for the suggestions so far, I should have given a little more > information in my initial post. The protein I'm working on is an Gram-Neg > Beta-Barrel about 100kDa, likely with 22 strands. > > I'm currently crystallising in bOG, although my next optimisation step is > to try a range of detergents. I'm using a refolding protocol which relies > heavily in LDAO (one of the only cost effective detergents for the volumes > I'm using), I refold, nickel purify (N-terminal tag, signal peptide > removed) , do a size exclusion step (S200), then detergent exchange into > bOG using a nickel column. The BOG concentration is obviously high 0.8-1%, > which might be causing the gels? > > The gels don't look like classic phase separation to me (from soluble > proteins that is, this is my first membrane protein), they are not > spherical, the tend to float or sit on the bottom and are semi-solid, > around 30-100uM diameter. These screens I've set up are classic screens for > membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't > dried up. The gels are strongly birefringent, but the drop is not. > Additionally the conditions in my initial screen which yielded crystals > seemed biased for the formation of the gels. > > Thanks again for the help, I feel I might have a long raod to optimisation > ahead of me. > > > Rhys > > ________________________________________ > From: Jim Fairman [fairman....@gmail.com] > Sent: 09 May 2013 20:58 > To: RHYS GRINTER > Cc: ccp4bb > Subject: Re: [ccp4bb] Membrane Protein Optimisation > > 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<mailto: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<mailto:fairman....@gmail.com> > jfair...@embios.com<mailto:jfair...@embios.com> >
