Jahan It sounds as though the protein crystallizes well, so microseeding (done the right way) is very likely to solve the problem. Make a strong seed stock with as much crystalline material as possible from several wells, including different hits if possible. Just mix them all together, but keep PEG conditions separate from salt conditions (or you will get two layers). Make a set of serial dilutions from "neat" up to 1 in 100,000 and freeze them at -80. You need to seed into *random screens*, so that you can pick up new conditions. Then you should optimize two or three new conditions by using the diluted seed stock. For example, if you estimate that there are 1000 crystals in the drop, you use the 1:1000 dilution.
For info and references see http://www.douglas.co.uk/mms.htm On 15 October 2012 23:01, Jahan Alikhajeh <ja...@graduate.org> wrote: > > Dear Friends, > > I am trying to crystalize a 70 kDa nasty protein but I got plate shape > crystals with high mosaicity and useless diffraction (up to 4A). > I tried to improve/optimize crystallization but either I got the same or > nothing. I tried seeding but I had so many crystals without any > improvement. Does anyone have better idea than routine optimization method > in the lab? Thanks in advance. > > Jahan -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
