Niks Seeding often works very well with needles. But start with seeding in RANDOM SCREENS, i.e. "rMMS"
References - Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007) 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 **Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 *Chatty article that I wrote for SerCat newsletter in 2009*: http://www.douglas.co.uk/SER-CAT09_1.html *A bit of theory*: http://www.douglas.co.uk/mms.htm *Procedure*: http://www.douglas.co.uk/MMS_proc.htm A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D66 (2010) 568-576. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444910005512 Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf On 24 July 2012 14:40, Niks <nik...@gmail.com> wrote: > > Dear All, > > I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. > Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). > I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. > I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. > > If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. > If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. > > Thanks very much > Nishant Varshney > PhD student, > National Chemical Laboratory,Pune,India > -- > "The most difficult phase of life is not when No one understands you;It is when you don't understand yourself" -- 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
