Skip to main content

Using Charge Ladders and Capillary Electrophoresis to Measure the Charge, Size, and Electrostatic Interactions of Proteins

  • Protocol
Capillary Electrophoresis of Proteins and Peptides

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 276))

Abstract

This chapter provides an overview of protein charge ladders—collections of protein derivatives that differ in charge—and capillary electrophoresis (CE). The combination of charge ladders and CE is a useful biophysical tool for measuring the net charge of proteins and the role of electrostatics in biochemical processes involving proteins. Methods to synthesize and analyze charge ladders by CE are described. Applications of charge ladders and CE to the simultaneous measurement of net charge and hydrodynamic radius of proteins are presented. Techniques for using charge ladders and CE to measure the role of interactions between charged groups on protein stability and ligand binding are also given. The power of this approach lies in the ability to isolate protein charge as an independent and measurable variable in the study of protein stability and function.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Collins, F. S., Patrinos, A., Jordan, E., Chakravarti, A., Gesteland, R., and Walters, L. (1998) New Goals for the U.S. Human Genome Project: 1998–2003. Science 282, 682–689.

    Article  PubMed  CAS  Google Scholar 

  2. Creighton, T. E. (1980) Counting integral numbers of amino acid residues per polypeptide chain. Nature 284, 487–489.

    Article  PubMed  CAS  Google Scholar 

  3. Gao, J., Gomez, F. A., Härter, R., and Whitesides, G. M. (1994) Determination of the effective charge of a protein in solution by capillary electrophoresis. Proc. Natl. Acad. Sci. USA 91, 12,027–12,030.

    Article  PubMed  CAS  Google Scholar 

  4. Colton, I. J., Anderson, J. R., Gao, J. M., Chapman, R. G., Isaacs, L., and Whitesides, G. M. (1997) Formation of protein charge ladders by acylation of amino groups on proteins. J. Amer. Chem. Soc. 119, 12,701–12,709.

    Article  CAS  Google Scholar 

  5. Carbeck, J. D., Colton, I. J., Gao, J. M., and Whitesides, G. M. (1998) Protein charge ladders, capillary electrophoresis, and the role of electrostatics in biomolecular recognition. Acc. Chem. Res. 31, 343–350.

    Article  CAS  Google Scholar 

  6. Carbeck, J. D. and Negin, R. S. (2001) Measuring the size and charge of proteins using protein charge ladders, capillary electrophoresis, and electrokinetic models of colloids. J. Amer. Chem. Soc. 123, 1252–1253.

    Article  CAS  Google Scholar 

  7. Hilser, V. J. and Freire, E. (1995) Quantitative analysis of conformational equilibrium using capillary electrophoresis: applications to protein folding. Anal. Biochem. 224, 465–485.

    Article  PubMed  CAS  Google Scholar 

  8. Colton, I. J., Carbeck, J. D., Rao, J., and Whitesides, G. M. (1998) Affinity capillary electrophoresis. A physical-organic tool for studying interactions in biomolecular recognition. Electrophoresis 19, 367–382.

    Article  PubMed  CAS  Google Scholar 

  9. Gao, J. M., Mammen, M., and Whitesides, G. M. (1996) Evaluating electrostatic contributions to binding with the use of protein charge ladders. Science 272, 535–537.

    Article  PubMed  CAS  Google Scholar 

  10. Caravella, J. A., Carbeck, J. D., Duffy, D. C., Whitesides, G. M., and Tidor, B. (1999) Long-range electrostatic contributions to protein-ligand binding estimated using protein charge ladders, affinity capillary electrophoresis, and continuum electrostatic theory. J. Amer. Chem. Soc. 121, 4340–4347.

    Article  CAS  Google Scholar 

  11. Negin, R. S. and Carbeck, J. D. (2002) Measurement of electrostatic interactions in protein folding with the use of protein charge ladders. J. Amer. Chem. Soc. 124, 2911–2916.

    Article  CAS  Google Scholar 

  12. Carbeck, J. D., Colton, I. J., Anderson, J. R., Deutch, J. M., and Whitesides, G. M. (1999) Correlations between the charge of proteins and the number of ionizable groups they incorporate: studies using protein charge ladders, capillary electrophoresis, and Debye-Hückel theory. J. Amer. Chem. Soc. 121, 10,671–10,679.

    Article  CAS  Google Scholar 

  13. Grossman, P. D. (1992) in Capillary Electrophoresis: Theory and Practice (Colburn, J. C., ed.), Academic, San Diego, CA.

    Google Scholar 

  14. Cordova, E., Gao, J. M., and Whitesides, G. M. (1997) Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins. Anal. Chem. 69, 1370–1379.

    Article  PubMed  CAS  Google Scholar 

  15. Henry, D. C. (1931) The cataphoresis of suspended particles. I. The equation of cataphoresis. Proc. Royal Soc. London 123, 106–129.

    Google Scholar 

  16. Menon, M. K. and Zydney, A. L. (2001) Protein charge ladders: a new technique for studying electrostatic interactions in ultrafiltration systems. J. Membr. Sci. 181, 179–184.

    Article  CAS  Google Scholar 

  17. Menon, M. K. and Zydney, A. L. (2000) Determination of effective protein charge by capillary electrophoresis: effects of charge regulation in the analysis of charge ladders. Anal. Chem. 72, 5714–5717.

    Article  PubMed  CAS  Google Scholar 

  18. Sharma, U., Negin, R. S., and Carbeck, J. D. (2003) Effects of cooperativity in proton binding on the net charge of proteins in charge ladders. J. Phys. Chem. B 107, 4653–4666.

    Article  CAS  Google Scholar 

  19. Basak, S. K., Velayudhan, A., and Ladisch, M. R. (1994) Characterization of buffers for electrokinetic separations. Appl. Biochem. Biotechnol. 44, 243–261.

    Article  CAS  Google Scholar 

  20. Ishihama, Y., Oda, Y., Asakawa, N., and Iwakura, M. (1997) Nano-scale monitoring of the thermally-induced unfolding of proteins using capillary electrophoresis with in-column incubation. Anal. Sciences 13, 931–938.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Sharma, U., Carbeck, J.D. (2004). Using Charge Ladders and Capillary Electrophoresis to Measure the Charge, Size, and Electrostatic Interactions of Proteins. In: Strege, M.A., Lagu, A.L. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology™, vol 276. Humana Press. https://doi.org/10.1385/1-59259-798-X:189

Download citation

  • DOI: https://doi.org/10.1385/1-59259-798-X:189

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-017-5

  • Online ISBN: 978-1-59259-798-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics