Skip to main content

Purification and Characterization of Human and Mouse Complement C3

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1100))

Abstract

Complement component C3 is the most abundant complement protein in plasma, central to all three complement activation pathways and essential to complement amplification. Thus, it is one of the most extensively studied complement proteins. This chapter describes the purification of C3 from human and mouse plasma using protein precipitation, followed by classical ion exchange chromatography and gel filtration. The biochemical and functional characteristics of the purified C3 are typically assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hemolysis assays. The hemolysis assay is a standard technique to assess complement activity monitoring the lysis of red blood cells.

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. de Bruijn MH, Fey GH (1985) Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci U S A 82:708–712

    Article  PubMed  Google Scholar 

  2. Lundwall A, Wetsel RA, Domdey H et al (1984) Structure of murine complement component C3. I. Nucleotide sequence of cloned complementary and genomic DNA coding for the beta chain. J Biol Chem 259:13851–13856

    PubMed  CAS  Google Scholar 

  3. Wetsel RA, Lundwall A, Davidson F et al (1984) Structure of murine complement component C3. II. Nucleotide sequence of cloned complementary DNA coding for the alpha chain. J Biol Chem 259:13857–13862

    PubMed  CAS  Google Scholar 

  4. Wetsel R (1995) Structure, function and cellular expression of complement anaphylatoxin receptors. Curr Opin Immunol 7:48–53

    Article  PubMed  CAS  Google Scholar 

  5. Nonaka M, Natsuume-Sakai S, Kaidoh T et al (1980) Characterization of allotypic determinants of murine complement C3. J Immunol 125:2025–2030

    PubMed  CAS  Google Scholar 

  6. Pangburn MK, Schreiber RD, Müller-Eberhard HJ (1981) Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3. J Exp Med 154:856–867

    Article  PubMed  CAS  Google Scholar 

  7. Amara U, Rittirsch D, Flierl M et al (2008) Interaction between the coagulation and complement system. Adv Exp Med Biol 632:71–79

    PubMed  CAS  Google Scholar 

  8. Brodbeck W, Mold C, Atkinson J et al (2000) Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack. J Immunol 165:3999–4006

    PubMed  CAS  Google Scholar 

  9. Lublin DM, Atkinson JP (1989) Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Immunol 7:35–58

    Article  PubMed  CAS  Google Scholar 

  10. Nicholson-Weller A, Wang C (1994) Structure and function of decay accelerating factor CD55. J Lab Clin Med 123:485–491

    PubMed  CAS  Google Scholar 

  11. Nicholson-Weller A, Burge J, Fearon DT et al (1982) Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J Immunol 129:184–189

    PubMed  CAS  Google Scholar 

  12. Pangburn MK, Schreiber RD, Muller-Eberhard HJ (1977) Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution. J Exp Med 146:257–270

    Article  PubMed  CAS  Google Scholar 

  13. Weiler JM, Daha MR, Austen KF et al (1976) Control of the amplification convertase of complement by the plasma protein beta1H. Proc Natl Acad Sci U S A 73:3268–3272

    Article  PubMed  CAS  Google Scholar 

  14. Morgan BP, Harris CL (1999) Complement regulatory proteins. Academic, San Diego, London

    Google Scholar 

  15. Fearon D (1980) Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J Exp Med 152:20–30

    Article  PubMed  CAS  Google Scholar 

  16. Ross G, Medof M (1985) Membrane complement receptors specific for bound fragments of C3. Adv Immunol 37:217–267

    Article  PubMed  CAS  Google Scholar 

  17. Ross G, Lambris J, Cain J et al (1982) Generation of three different fragments of bound C3 with purified factor I or serum. I. Requirements for factor H vs. CR1 cofactor activity. J Immunol 129:2051–2060

    PubMed  CAS  Google Scholar 

  18. Foley S, Li B, Dehoff M et al (1993) Mouse Crry/p65 is a regulator of the alternative pathway of complement activation. Eur J Immunol 23:1381–1384

    Article  PubMed  CAS  Google Scholar 

  19. Kim YU, Kinoshita T, Molina H et al (1995) Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein. J Exp Med 181:151–159

    Article  PubMed  CAS  Google Scholar 

  20. van Lookeren Campagne M, Wiesmann C, Brown EJ (2007) Macrophage complement receptors and pathogen clearance. Cell Microbiol 9:2095–2102

    Article  PubMed  Google Scholar 

  21. Harrison R (1996) Purification, assay, characterization of complement proteins from plasma in handbook of experimental immunology. In: Leonore A. Herzenberg (ed) Cell surface and messenger molecules of the immune system, Donald M. Weir/co-editors, vol 2, 5th edn, Caroline Blackwell. edn, Blackwell Science, Cambridge

    Google Scholar 

  22. Koch C, Skjødt K, Laursen I (1985) A simple immunoblotting method after separation of proteins in agarose gel. J Immunol Methods 84:271–278

    Article  PubMed  CAS  Google Scholar 

  23. Natsuume-Sakai S, Moriwaki K, Amano S et al (1979) Allotypes of C3 in laboratory and wild mouse distinguished by alloantisera. J Immunol 123:216–221

    PubMed  CAS  Google Scholar 

  24. Ruseva MM, Hughes TR, Donev RM et al (2009) Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury. Mol Immunol 46:803–811

    Article  PubMed  CAS  Google Scholar 

  25. Morgan BP (2000) Complement methods and protocols. Humana, New York

    Book  Google Scholar 

  26. van Dijk H, Rademaker PM, Willers JM (1980) Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes. J Immunol Methods 39:257–268

    Article  PubMed  Google Scholar 

  27. Morgan BP (2000) Measurement of complement hemolytic activity, generation of complement-depleted sera, and production of hemolytic intermediates. Methods Mol Biol 150:61–71

    PubMed  CAS  Google Scholar 

  28. Heurich M, Martínez-Barricarte R, Francis NJ et al (2011) Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. Proc Natl Acad Sci U S A 108:8761–8766

    Article  PubMed  CAS  Google Scholar 

  29. Isenman DE, Kells DI, Cooper NR et al (1981) Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties. Biochemistry 20:4458–4467

    Article  PubMed  CAS  Google Scholar 

  30. Lachmann PJ (2010) Preparing serum for functional complement assays. J Immunol Methods 352:195–197

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The methods and protocols described in this chapter were derived and optimized in the laboratory of Dr. Claire L Harris and Prof. B Paul Morgan (School of Medicine, Cardiff University, United Kingdom).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Ruseva, M.M., Heurich, M. (2014). Purification and Characterization of Human and Mouse Complement C3. In: Gadjeva, M. (eds) The Complement System. Methods in Molecular Biology, vol 1100. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-724-2_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-724-2_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-723-5

  • Online ISBN: 978-1-62703-724-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics