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Tricine–SDS-PAGE

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Abstract

Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine–SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1–2 d.

*Note: In the version of the article initially published online, the words “Gel buffer (3x)” were missing in the table on page 18.  The error has been corrected in all versions of the article.

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Figure 1: Comparison of Tricine–SDS-PAGE and Laemmli–SDS-PAGE.
Figure 2: Tricine–SDS-PAGE of marker proteins using various types of gel.

Change history

  • 10 August 2006

    In the version of the article initially published online, the words “Gel buffer (3x)” were missing in the table on page 18. The error has been corrected in all versions of the article.

References

  1. 1

    Laemmli, U.K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    CAS  Article  Google Scholar 

  2. 2

    Schägger, H. & von Jagow, G. Tricine–sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1–100 kDalton. Anal. Biochem. 166, 368–379 (1987).

    Article  Google Scholar 

  3. 3

    Schägger, H. SDS electrophoresis techniques. in Membrane Protein Purification and Crystallization. A Practical Guide 2nd edn. (eds. Hunte, C., von Jagow, G. & Schägger, H.) 4.85–4.103 (Academic, San Diego, California, 2003).

    Google Scholar 

  4. 4

    Hjerten, S. Chromatographic separation according to size of macromolecules and cell particles on columns of agarose suspensions. Arch. Biochem. Biophys. 99, 466–475 (1962).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Rabilloud, T. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13, 429–439 (1992).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Blum, H., Beier, H. & Gross, H.J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99 (1987).

    CAS  Article  Google Scholar 

  7. 7

    Rais, I., Karas, M. & Schägger, H. Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 4, 2567–2571 (2004).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Renart, J., Reiser, J. & Stark, G.R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76, 3116–3120 (1979).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Kyhse-Andersen, J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10, 203–209 (1984).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Beisiegel, U. Protein blotting. Electrophoresis 7, 1–18 (1986).

    CAS  Article  Google Scholar 

  11. 11

    Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4356 (1979).

    CAS  Article  Google Scholar 

  12. 12

    Acin-Perez, R. et al. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell 13, 805–815 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Schägger, H. et al. Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J. Biol. Chem. 279, 36349–36353 (2004).

    Article  PubMed  Google Scholar 

  14. 14

    Carrozzo, R. et al. Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders. Ann. Neurol. 59, 265–275 (2006).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Griffon, N. et al. Molecular determinants of glycine receptor subunit assembly. EMBO J. 18, 4711–4721 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Dietmeyer, K. et al. Tom 5 functionally links mitochondrial preprotein receptors to the general import pore. Nature 388, 195–200 (1997).

    Article  Google Scholar 

  17. 17

    Jänsch, L., Kruft, V., Schmitz, U.K. & Braun, H.-P. Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants. J. Biol. Chem. 273, 17251–17257 (1998).

    Article  PubMed  Google Scholar 

  18. 18

    Vahsen, N. et al. AIF deficiency compromises oxidative phosphorylation. EMBO J. 23, 4679–4689 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Schägger, H., Cramer, W.A. & von Jagow, G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217, 220–230 (1994).

    Article  PubMed  Google Scholar 

  20. 20

    Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R.A. & Schägger, H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J. 17, 7170–7178 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Schägger, H. & Pfeiffer, K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783 (2000).

    PubMed  PubMed Central  Google Scholar 

  22. 22

    Pfeiffer, K. et al. Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol. Chem. 278, 52873–52880 (2003).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Schägger, H. & von Jagow, G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231 (1991).

    Article  PubMed  Google Scholar 

  24. 24

    Schägger, H. Blue native electrophoresis. in Membrane Protein Purification and Crystallization. A Practical Guide 2nd edn. (eds. Hunte, C., von Jagow, G. & Schägger, H.) 5.105–5.130 (Academic, San Diego, California, 2003).

    Google Scholar 

  25. 25

    Wittig, I. & Schägger, H. Advantages and limitations of clear native polyacrylamide gel electrophoresis. Proteomics 5, 4338–4346 (2005).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Wittig, I., Braun, H.-P. & Schägger, H. Blue-native electrophoresis. Nat. Protocols (in the press).

  27. 27

    Studier, F.W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237–248 (1973).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Swank, R.T. & Munkres, K.D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 39, 462–477 (1971).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 628, Project P13.

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Correspondence to Hermann Schägger.

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Schägger, H. Tricine–SDS-PAGE. Nat Protoc 1, 16–22 (2006). https://doi.org/10.1038/nprot.2006.4

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