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Protein Blotting by the Semidry Method

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The Protein Protocols Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

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Abstract

Protein blotting, also known as Western blotting, refers to the transfer of proteins to an immobilizing membrane. The most widely used blotting method is the electrophoretic transfer of resolved proteins from a polyacrylamide gel to a nitrocellulose or polyvinylidene difluoride (PVDF) sheet and is often referred to as “Western blotting.” Electrophoretic transfer uses the driving force of an electric field to elute proteins from gels and to immobilize them on a matrix. This method is fast, efficient and maintains the high resolution of the protein pattern (1). There are currently two main configurations of electroblotting apparatus: (1) tanks of buffer with vertically placed wire (see Chapter 58) or plate electrodes and (2) semidry transfer with flat-plate electrodes.

For semidry blotting, the gel and membrane are sandwiched horizontally between two stacks of buffer-wetted filter papers which are in direct contact with two closely spaced solid-plate electrodes. The name semidry refers to the limited amount of buffer which is confined to the stacks of filter paper. Semidry blotting requires considerably less buffer than the tank method, the transfer from single gels is simpler to set up, it allows the use of multiple transfer buffers (i.e different buffers in the cathode and anode electrolyte stacks) and it is reserved for rapid transfers because the amount of buffer is limited and the use of external cooling is not possible. Nevertheless, both techniques have a high efficacy and the choice between the two types of transfer is a matter of preference.

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References

  1. Garfin, D. E. and Bers, G. (1982) Basic aspects of protein blotting, in Protein blotting (Baldo B.A. and Tovey, E. R. eds), Karger, Basel, Switzerland, pp.5–42.

    Google Scholar 

  2. Kurien, B. T. and Scotfield R. H. (2006) Western blotting. Methods 38, 283–293.

    Article  CAS  PubMed  Google Scholar 

  3. Eckerskorn, C., Strupat, K., Schleuder, D., Hochstrasser, D. F., Sanchez, J. C., Lottspeich, F., and Hillenkamp, F. (1997) Analysis of proteins by direct scanning-infrared-MALDI mass spectrometry after 2-D PAGE separation and electroblot-ting. Anal. Chem. 69, 2888–2892.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Gershoni, J. M. and Palade, G. E. (1983) Protein blotting: principles and applications. Anal. Biochem. 131, 1–15.

    Article  CAS  PubMed  Google Scholar 

  6. Gravel P. (2007) Protein blotting, in Protein and Cell Biomethods Handbook, 2nd ed. (Walker, J. M. and Rapley, R., eds), Humana Press Inc, Totowa, NJ.

    Google Scholar 

  7. Wilkins, M. R. and Gooley, A. A. (1997) Protein identification in proteome projects, in Proteomic Research: New Frontiers in Functional Genomics (Principles and Practice) (Wilkins, M. R., Williams, K. L., Appel, R. D., and Hochstrasser, D.F., eds), Springer Verlag, Berlin, pp. 35–64.

    Chapter  Google Scholar 

  8. Gravel, P., Golaz, O., Walzer, C., Hochstrasser, D.F., Turler, H., and Balant, L.P. (1994) Analysis of glycoproteins separated by two-dimensional gel electrophoresis using lectin blotting revealed by chemiluminescence. Anal. Biochem. 221, 66–71.

    Article  CAS  PubMed  Google Scholar 

  9. Sanchez, J. C., Ravier, F., Pasquali, C., Frutiger, S., Bjellqvist, B., Hochstrasser, D. F., and Hughes, G.J. (1992) Improving the detection of proteins after transfer to polyvinylidene difluoride membranes. Electrophoresis 13, 715–717.

    Article  CAS  PubMed  Google Scholar 

  10. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Erickson, P. F., Minier, L. N., and Lasher, R. S. (1982) Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens. J. Immunol. Methods 51, 241–249.

    Article  CAS  PubMed  Google Scholar 

  12. Dunn, S. D. (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal. Biochem. 157, 144–153.

    Article  CAS  PubMed  Google Scholar 

  13. Bjerrum, O. J. and Schafer-Nielsen, C. (1986). Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus in “Electrophoresis 1986” (Dunn, MJ, ed). VCH, Weinheim, pp. 315–227..

    Google Scholar 

  14. Gershoni, J. M. and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124, 396–405.

    Article  CAS  PubMed  Google Scholar 

  15. Jin, Y. and Cerletti, N. (1992) Western blotting of transforming growth factor b2. Optimization of the electrophoretic transfer. Appl. Theor. Electrophoresis 3, 85–90.

    CAS  Google Scholar 

  16. Matsudaira, P. J. (1987) Sequence from picomole quantities of proteins elec-troblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 21, 10035–10038.

    Google Scholar 

  17. Svoboda, M., Meuris, S., Robyn, C., and Christophe, J. (1985) Rapid electrotrans-fer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode. Anal. Biochem. 151, 16–23.

    Article  CAS  PubMed  Google Scholar 

  18. Kyhse-Andersen, J. J. (1984) 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.

    Article  CAS  PubMed  Google Scholar 

  19. Laurière, M. (1993) A semidry electroblotting system efficiently transfers both high and low molecular weight proteins separated by SDS-PAGE. Anal. Biochem. 212, 206–211.

    Article  PubMed  Google Scholar 

  20. Zeng, C., Suzuki, Y., and Alpert, E. (1990) Polyethylene glycol significantly enhances the transfer of membrane immunoblotting. Anal. Biochem. 189, 197–201.

    Article  CAS  PubMed  Google Scholar 

  21. Vachereau, A. (1989) Transparency of nitrocellulose membranes with Triton X-114. Electrophoresis 10, 524–527.

    Article  CAS  PubMed  Google Scholar 

  22. Alimi, E., Martinage, A., Sautière, P., and Chevaillier, P. (1993) Electroblotting proteins onto carboxymethylcellulose membranes for sequencing. BioTechniques 15, 912–917.

    CAS  PubMed  Google Scholar 

  23. Hauri, H.P. and Bucher, K. (1986) Immunoblotting with monoclonal antibodies: importance of the blocking solution. Anal. Biochem. 159, 386–389.

    Article  CAS  PubMed  Google Scholar 

  24. Rohringer, R. and Holden, D. W. (1985) Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes. Anal. Biochem. 144, 118–127.

    Article  CAS  PubMed  Google Scholar 

  25. Saravis, C. A. (1984) Improved blocking of nonspecific antibody binding sites on nitrocellulose membranes. Electrophoresis 5, 54–55.

    Article  CAS  Google Scholar 

  26. Batteiger, B., Newhall, W. J., and Jones, R. B. (1982) The use of tween-20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55, 297–307.

    Article  CAS  PubMed  Google Scholar 

  27. Haycock, J. W. (1993) Polyvinylpyrrolidone as a blocking agent in immunochemical studies. Anal. Biochem. 208, 397–399.

    Article  CAS  PubMed  Google Scholar 

  28. Hoffman, W. L. and Jump, A. A. (1986) Tween-20 removes antibodies and other proteins from nitrocellulose. J. Immunol. Methods 94, 191–196.

    Article  CAS  PubMed  Google Scholar 

  29. Gravel, P., Sanchez, J. C., Walzer, C., Golaz, O., Hochstrasser, D. F., Balant, L. P., Hughes, G. J., Garcia-Sevilla, J., and Guimon, J. Human blood platelet protein map established by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 16, 1152–1159.

    Google Scholar 

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Authors and Affiliations

  1. Triskel Integrated Services SA, Geneva, Switzerland

    Patricia Gravel

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  1. Patricia Gravel
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Editors and Affiliations

  1. School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK

    John M. Walker

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Gravel, P. (2009). Protein Blotting by the Semidry Method. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_59

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  • DOI: https://doi.org/10.1007/978-1-59745-198-7_59

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-474-6

  • Online ISBN: 978-1-59745-198-7

  • eBook Packages: Springer Protocols

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