Skip to main content

Efficient Macromolecular Crystallization Using Microfluidics and Randomized Design of Screening Reagents

  • Protocol
Structural Proteomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 426))

  • 3670 Accesses

Microfluidic technologies enable a relatively new approach to macromolecular crystallization, but offer several significant advantages over more traditional techniques. Microfluidic devices provide significant savings in the amount of material required to complete a set of experiments, although recent innovations with vapor diffusion and microbatch methods have also greatly reduced their material requirements. When compared with these other methods, microfluidic approaches still consume 5–100× less material. In addition, comparisons in one set of experiments suggest that microfluidic free-interface diffusion may also offer substantially higher success rates than sitting drop vapor diffusion. Microfluidic methods also provide opportunities for experimental strategies involving testing multiple samples in parallel. When combined with randomized design of screening reagents, microfluidic devices provide a highly efficient method for sampling crystallization space. Commercial microfluidic crystal lization chips have been in circulation for a number of years now and stable protocols for their use, tips and tricks, and data on their success and failure are now available.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hansen, C. L., Skordalakes, E., Berger, J. M., and Quake, S. R. (2002) A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc. Natl. Acad. Sci. USA 99, 16531–16536.

    Article  CAS  PubMed  Google Scholar 

  2. Zheng, B., Roach, L. S., Rustem, F., and Ismagilov, R. F. (2003) Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets. J. Am. Chem. Soc. 125, 11170–11171.

    Article  CAS  PubMed  Google Scholar 

  3. Yamada, M., Sasaki, C., Isomura, T., and Seki, M. (2003) Microfluidic reactor array for high-throughput screening of protein crystallization conditions. 7th International Conference on Miniaturized Chemical and Biochemical Analysts Systems October 5–9, 2003, Squaw Valley, CA.

    Google Scholar 

  4. Chao, W.-C., Collins, J., Bachman, M., Lia, G. P., and Lee, A. P. (2004) Droplet arrays in microfluidic channels for combinatorial screening assays. Solid-State Sensor, Actuator and Microsystems Workshop Hilton Head Island, SC, June 6–10, 2004.

    Google Scholar 

  5. Segelke, B. W., Schafer, J., Coleman, M. A., Lekin, T. P., Toppani, D., Skowronek, K. J., Kantardjieff, K. A., and Rupp, B. (2004) Laboratory scale structural genomics. J. Struct. Funct. Genom. 5, 147–157.

    Article  CAS  Google Scholar 

  6. D'Arcy, A., Sweeney, A. M., and Haber, A. (2004) Practical aspects of using the microbatch method in screening conditions for protein crystallization. Methods 34, 323–328.

    Article  PubMed  Google Scholar 

  7. D'Arcy, A., MacSweeney, A., and Habera, A. (2004) Modified microbatch and seeding in protein crystallization experiments. J. Synchrotron Radiat. 11, 24–26.

    Article  PubMed  Google Scholar 

  8. Luft, J. R., Collins, R. J., Fehrman, N. A., Lauricella, A. M., Veatch, C. K., and DeTitta, G. T. (2003) A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol. 142, 170–179.

    Article  CAS  PubMed  Google Scholar 

  9. Stevens, R. C. (2000) High-throughput protein crystallization. Curr. Opin. Struct. Biol. 10, 558–563.

    Article  CAS  PubMed  Google Scholar 

  10. Hansen, C. L., Sommer, M. O. A., and Quake, S. R. (2004) Systematic investigation of protein phase behavior with a microfluidic formulator. Proc. Natl. Acad. Sci. USA 101, 14431–14436.

    Article  CAS  PubMed  Google Scholar 

  11. Chen, D. L., Gerdts, C. J., and Ismagilov, R. F. (2005) Using microfluidics to observe the effect of mixing on nucleation of protein crystals. J. Am. Chem. Soc. 127, 9672–9673.

    Article  CAS  PubMed  Google Scholar 

  12. Segelke, B. W. (2001) Efficiency analysis of screening protocols used in protein crystallization. J. Crystal Growth 232, 553–562.

    Article  CAS  Google Scholar 

  13. Derewenda, Z. S., and Vekilov, P. G. (2006) Entropy and surface engineering in protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 62, 116–124.

    Article  PubMed  Google Scholar 

  14. Rowland, P., Blaney, F. E., Smyth, M. G., Jones, J. J., Leydon, V. R., Oxbrow, A. K., Lewis, C. J., Tennant, M. G., Modi, S., Eggleston, D. S., Chenery, R. J., and Bridges, A. M. (2006) Crystal structure of human cytochrome P450 2D6. J. Biol. Chem. 281, 7614–7622.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was carried out at the Lawrence Livermore National Lab and was performed under the auspices of the U.S. Department of Energy by University of California, Lawrence Livermore National Laboratory under contract W-7405-Eng-48. The authors thank Kevin Farrell and Yong Yi for comments on the manuscript. The authors also thank the TB structural genomics consortium for support for some of the work described.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

May, A.P., Segelke, B.W. (2008). Efficient Macromolecular Crystallization Using Microfluidics and Randomized Design of Screening Reagents. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_25

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-058-8_25

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-809-6

  • Online ISBN: 978-1-60327-058-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics