Nucleation and seeding in protein crystallization - Hampton Research

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Separating nucleation and growth (~28% PEG 8000, 0.1 M sodium cacodylate pH 6.5) 25% PEG 8000, 0.1 M calcium acetate, 0.1 M sodium cacodylate pH 6.5 - Ca + Ca All attempts to grow the new crystal form under these conditions by de novo nucleation (without transfer of microseeds) were unsuccessful. 28
Microseed Matrix Seeding: preparation of seeds • Seed stock preparation: • Select best crystals possible • Crush crystals in their reservoir solution using Hampton tools • Transfer to Hampton seed bead and vortex for at least 3 minutes or place on Thermoshaker 10“ on 2“ off for 10-30 mins • Store at -80°C
Page 1 and 2: Nucleation and seeding in protein c
Page 3 and 4: Growing protein crystals We have 2
Page 5 and 6: Classes of proteins (an oversimplif
Page 7 and 8: Protein very soluble, no nucleation
Page 9 and 10: Nucleation • The growth of protei
Page 11 and 12: Drop set up (mix) Mixing drops
Page 13 and 14: Drop size • D. G. Georgieva, M. E
Page 15 and 16: Different seeding methods
Page 17 and 18: Different seeding methods: Seed bea
Page 19 and 20: Different seeding methods: Heteroge
Page 21 and 22: The first artifial snow crystals
Page 23 and 24: Cause and effect/proof of concept
Page 25 and 26: Other nucleation agents • McPhers
Page 27: Important publication Acta Cryst. (
Page 31 and 32: Screening for initial crystallizati
Page 33 and 34: Microcrystalline droplets/phase sep
Page 35 and 36: You may generate more false positiv
Page 37 and 38: Using UV to identify protein crysta
Page 39 and 40: Example of confirmed screen hit
Page 41 and 42: CYS protease • Starting crystals
Page 43 and 44: Metallo Protease Only one crystal g
Page 45 and 46: Eliminate twinning Result: Twinning
Page 47 and 48: Fab complexes, another powerful cry
Page 49 and 50: Conclusions and questions • In pr
Page 51: Backup

Nucleation and seeding in protein crystallization - Hampton Research

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