Advertisement

Analysis of Protein Complexes by 2D Blue Native/SDS–PAGE and Antibody-Shift Assay

  • Dong Yang
  • Xinyu Deng
  • Ying JiangEmail author
  • Fuchu He
Protocol
Part of the Neuromethods book series (NM, volume 57)

Abstract

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful strategy for the separation of native multiprotein complex (MPC). Combined with other techniques, such as SDS–PAGE, immunoblotting, mass spectrometry, and antibody-shift assay, BN-PAGE has been widely used in MPC research. Here, we describe the materials and methods for the basic techniques mentioned above and provide the extensive practical advice and potential pitfalls of BN-PAGE.

Key words

BN-PAGE Multiprotein complex Antibody-shift assay 

Notes

Acknowledgments

This work was partially supported by National Natural Science Foundation of China for Creative Research Groups (30621063) and International Scientific Collaboration Program (2009DFB33070)

References

  1. 1.
    Schägger H, von Jagow G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199, 223–231.PubMedCrossRefGoogle Scholar
  2. 2.
    Wittig I, Braun HP, Schägger H. (2006) Blue native PAGE. Nat Protoc.1, 418–428.Google Scholar
  3. 3.
    Schägger, H., Cramer, W. A., von Jagow, G. (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane ­protein complexes by twodimensional native electrophoresis. Anal. Biochem. 217, 220–230.PubMedCrossRefGoogle Scholar
  4. 4.
    Aufurth S, Schagger H, Muller V. (2000) Identification of subunits a, b, and c1 from Acetobacterium woodii Na+−F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer. J Biol Chem 275, 33297–33301.PubMedCrossRefGoogle Scholar
  5. 5.
    Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP. (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol 127, 1694–1710.PubMedCrossRefGoogle Scholar
  6. 6.
    Camacho-Carvajal MM, Wollscheid B, Aebersold R, Steimle V, Schamel WW. (2004) Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach. Mol Cell Proteomics 3, 176–182.PubMedGoogle Scholar
  7. 7.
    Liu K, Qian L, Wang J, Li W, Deng X, Chen X, et al. (2009) Two-dimensional blue native/SDS–PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells. Mol Cell Proteomics 8, 495–505.PubMedCrossRefGoogle Scholar
  8. 8.
    Hellwig S, Schamel WW, Pflugfelder U, Gerlich B, Weltzien HU. (2005) Differences in pairing and cluster formation of T cell receptor alpha- and beta-chains in T cell clones and fusion hybridomas. Immunobiology 210, 685–694.PubMedCrossRefGoogle Scholar
  9. 9.
    Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, et al. (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J 17, 3016–3028.PubMedCrossRefGoogle Scholar
  10. 10.
    Nicke A, Kerschensteiner D, Soto F. (2005) Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits. J Neurochem 92, 925–33.PubMedCrossRefGoogle Scholar
  11. 11.
    Hedman E, Widen C, Asadi A, Dinnetz I, Schroder WP, Gustafsson JA, et al. (2006) Proteomic identification of glucocorticoid receptor interacting proteins. Proteomics 6, 3114–3126.PubMedCrossRefGoogle Scholar
  12. 12.
    Deng X, Li W, Chen N, Sun Y, Wei H, Jiang Y, et al. (2009) Exploring the priming mechanism of liver regeneration: proteins and protein complexes. Proteomics 9, 2202–2216.PubMedCrossRefGoogle Scholar
  13. 13.
    Evin G, Canterford LD, Hoke DE, Sharples RA, Culvenor JG, Masters CL. (2005) Transition-state analogue gamma-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex. Biochemistry 44, 4332–4341.PubMedCrossRefGoogle Scholar
  14. 14.
    Culvenor JG, Ilaya NT, Ryan MT, Canterford L, Hoke DE, Williamson NA, et al. (2004) Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations. Eur J Biochem 271, 375–385.PubMedCrossRefGoogle Scholar
  15. 15.
    Deng XY, Li WR, Sun YW, Wei HD, Jiang Y, He FC. (2009) Exploring rat plasmatic proteomes: what triggered the liver regeneration? Protein Pept Lett 16, 698–705.PubMedCrossRefGoogle Scholar
  16. 16.
    Schamel, W. W. A., Reth, M. (2000) Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity 13, 5–14.PubMedCrossRefGoogle Scholar
  17. 17.
    Wittig I, Schagger H. (2008) Features and applications of blue-native and clear-native electrophoresis. Proteomics 8, 3974–3990.PubMedCrossRefGoogle Scholar
  18. 18.
    Zhang P, Battchikova N, Paakkarinen V, Katoh H, Iwai M, Ikeuchi M, et al. (2005) Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1. Biochem J 390(Pt 2), 513–520.PubMedGoogle Scholar
  19. 19.
    Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M. (2006) A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis. Electrophoresis 27, 3306–3321.PubMedCrossRefGoogle Scholar
  20. 20.
    Schägger H, Pfeiffer K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19, 1777–1783.PubMedCrossRefGoogle Scholar
  21. 21.
    Wittig I, Schagger H. (2005) Advantages and limitations of clear-native PAGE. Proteomics 5, 4338–4346.PubMedCrossRefGoogle Scholar
  22. 22.
    Devreese B, Vanrobaeys F, Smet J, Van Beeumen J, Van Coster R. (2002) Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis. Electrophoresis 23, 2525–2533.PubMedCrossRefGoogle Scholar
  23. 23.
    Millar AH, Eubel H, Jansch L, Kruft V, Heazlewood JL, Braun HP. (2004) Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits. Plant Mol Biol 56, 77–90.PubMedCrossRefGoogle Scholar
  24. 24.
    Reifschneider NH, Goto S, Nakamoto H, Takahashi R, Sugawa M, Dencher NA, et al. (2006) Defining the mitochondrial proteomes from five rat organs in a physiologically ­significant context using 2D blue-native/SDS–PAGE. J Proteome Res 5, 1117–1132.PubMedCrossRefGoogle Scholar
  25. 25.
    Werhahn W, Braun HP. (2002) Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis. Electrophoresis 23, 640–646.PubMedCrossRefGoogle Scholar
  26. 26.
    Reisinger V, Eichacker LA. (2007) How to analyze protein complexes by 2D blue native SDS–PAGE. Proteomics 7 Suppl 1, 6–16.PubMedCrossRefGoogle Scholar
  27. 27.
    Heinemeyer J, Scheibe B, Schmitz UK, Braun HP. (2009) Blue native DIGE as a tool for comparative analyses of protein complexes. J Proteomics 72, 539–544.PubMedCrossRefGoogle Scholar
  28. 28.
    Poetsch A, Neff D, Seelert H, Schagger H, Dencher NA. (2000) Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis. Biochim Biophys Acta 1466, 339–349.PubMedCrossRefGoogle Scholar
  29. 29.
    Seelert H, Dencher NA, Muller DJ. (2003) Fourteen protomers compose the oligomer III of the proton-rotor in spinach chloroplast ATP synthase. J Mol Biol 333, 337–44.PubMedCrossRefGoogle Scholar
  30. 30.
    Wittig I, Karas M, Schagger H. (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6, 1215–1225.PubMedCrossRefGoogle Scholar
  31. 31.
    Meyer B, Wittig I, Trifilieff E, Karas M, Schägger H. (2007) Identification of two proteins ­associated with mammalian ATP synthase. Mol Cell Proteomics 6, 1690–1699.PubMedCrossRefGoogle Scholar
  32. 32.
    Wittig I, Carrozzo R, Santorelli FM, Schagger H. (2007) Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines. Electrophoresis, 28, 3811–3820.PubMedCrossRefGoogle Scholar
  33. 33.
    Henderson NS, Nijtmans LG, Lindsay JG, Lamantea E, Zeviani M, Holt IJ. (2000) Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis.Electrophoresis 21, 2925–2931.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.State Key Laboratory of ProteomicsBeijing Proteome Research Center, Beijing Institute of Radiation MedicineBeijingChina

Personalised recommendations