You did check on a gel that they are indeed your protein ? If you have sufficient amounts available try digesting it with various proteases and see if you can identify a stable fragment.
A less radical approach, which might not be accessible to you, you could screen your protein for alternative buffer conditions using DSF and then pick a condition under which it seems to be very stable according to its melting temperature in the buffer. You've spared us the details of your purification procedure, maybe a polishing step at the end with a SEC might do wonders. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 26, 2010, at 4:23 PM, Matthew Bratkowski wrote: > Hello. > > I have obtained disk shaped crystals of a protein that I am working on. I > got hits in about 10 different conditions, with a few common precipitants and > pHs, and I have optimized two conditions so far. In the optimized > conditions, the crystals appear overnight, usually surrounded by or hiding > under heavy precipitant. Under the best conditions, I get what I would > describe as single disks, some of which are of decent size and very round, > that rotate light very well. Sub-optimal conditions can give small to large > crystal clusters. I shot the large disk crystals grown from one conditions > at the synchrotron. but they do not diffract. > > I was wondering if anyone had any advice about optimizing these crystals in > order to get them to diffract better? As mentioned before, I have only tried > optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), > but crystals from all of the hits look the same: always round disks or disk > clusters. This leads me to believe that optimized conditions of the other > hits will produce similar results as before. Would it be worthwhile to try > optimizing these conditions as well? I have also tried seeding, which just > produces a lot of clusters, and an additive screen. Some of the additives > help to produce larger crystals, but again I always get single or disk > clusters. > > Any advice would be helpful. > > Thanks, > Matt > >
