apart from optimising the crystallisation conditions, it might be
worth optimising the protein preparation or do some limited
proteolysis - or even express short N-terminal and/or C-terminal
deletions.
Mark
Quoting Patrick Shaw Stewart:
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
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es
