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Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London:

Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London: Professor David M. Blow, Patrick Shaw Stewart, Dennis Maeder, Naomi Chayen Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Vaughan Mills, James Smith.

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Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London:

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  1. Introduction to microbatch protein crystallization Patrick Shaw Stewart Imperial College, London: Professor David M. Blow, Patrick Shaw Stewart, Dennis Maeder, Naomi Chayen Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Vaughan Mills, James Smith

  2. What is microbatch crystallization? • Crystallization in small drops under oil

  3. What is microbatch crystallization? • Crystallization in small drops under oil • 100 + 100 nl to 1+1 µl

  4. What is microbatch crystallization? • Crystallization in small drops under oil • 100 + 100 nl to 1+1 µl • The oil prevents evaporation

  5. Why is microbatch a good idea?

  6. Why is microbatch a good idea? • Easy

  7. Why is microbatch a good idea? • Easy • Gives better crystals in many cases – especially in screening

  8. Why is microbatch a good idea? • Easy • Gives better crystals in many cases – especially in screening • It doesn’t matter if the security guard at the airport puts it through the x-ray machine upside down

  9. Why is microbatch a good idea? • Easy • Gives better crystals in many cases – especially in screening • It doesn’t matter if the security guard at the airport puts it through the x-ray machine upside down • Cheap!

  10. Microbatch crystallization Volume of well - 12 microlitres

  11. Microbatch crystallization Volume of drop - 0.2 to 2 microlitres

  12. Microbatch crystallization (2-bore) microtip Oil Sample

  13. Microbatch crystallization

  14. Microbatch crystallization

  15. Microbatch optimization – print out

  16. Microbatch optimization – print out

  17. ORYX 6 crystallization system

  18. Liquid-handling channel Motorized Hamilton gas-tight syringe (water) X 5 to microtip

  19. ORYX 6 crystallization system

  20. Large-volume tip for oil

  21. Left-hand tip: 2-bore Microtip – screening 5-bore Microtip – optimization

  22. End of a 5-bore microtip 0.15 mm 0.9 mm

  23. Microbatch screening

  24. 2-bore Microtip – screening

  25. Microbatch screening – dispensing cycle Target plate Screening solutions

  26. Sitting Drop - preparation Air bubble • Suck up protein required for experiment + 0.25 µl • Suck air bubble in second bore – for transfer Protein slug Air bubble

  27. Microbatch screening – “sip and spit” • Pick up 100 nl screening solution (1)

  28. Microbatch screening – “sip and spit” • Pick up 100 nl screening solution • Transfer to microbatch drop and add protein (1) (2)

  29. Microbatch optimization

  30. 5-bore Microtip – optimization

  31. Microbatch optimization – dispensing cycle • Dispense five solutions together (1)

  32. Microbatch optimization – dispensing cycle • Dispense five solutions together • Oil (2) oil

  33. Central Composite design

  34. Sitting Drop – dispensing cycle • Rinse in reservoir • Move sideways and pick up clean solution • Dispense solution and protein (3) (1) (2)

  35. Phase diagram of a protein precipitate [Protein] clear [Precipitant]

  36. Phase diagram of a protein precipitate nucleation [Protein] clear [Precipitant]

  37. precipitate nucleation [Protein] metastable zone clear [Precipitant] Phase diagram of a protein

  38. Phase diagram of a protein p n [Protein] m.z. Vapor diffusion c [Precipitant]

  39. Phase diagram of a protein p n Microbatch [Protein] m.z. v.d. c [Precipitant]

  40. Phase diagram of a protein p n M.B.(paraffin) [Protein] m.z. v.d.. M.B.(par./si.) c [Precipitant]

  41. Phase diagram of a protein p n M.B.(paraffin) OPTIMIZATION [Protein] m.z. v.d. M.B.(par./si.) SCREENING [Precipitant]

  42. What % of protein should you use? Microbatch with Si. / Par.: n [Protein] m.z. Precipitant saturated [Precipitant]

  43. What % of protein should you use? Microbatch with Si. / Par.: n [Protein] Protein stock m.z. 50% Precipitant saturated Precipitant stock [Precipitant]

  44. What % of protein should you use? Microbatch with Si. / Par.: n [Protein] Protein stock m.z. 66% 50% Precipitant saturated Precipitant stock [Precipitant]

  45. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm

  46. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm A. D’Arcy, G.E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000.

  47. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm A. D’Arcy, G.E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt

  48. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm A. D’Arcy, G.E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

  49. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm A. D’Arcy, G.E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

  50. OPTIMIZATION: about 50:50 • In microbatch, there tends to be more precipitation initially; this may result in more nucleation

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