Skip to main content
SpringerLink
Log in

Protein (western) blotting

  • Review
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The different steps involved in protein (Western) blotting and subsequent analysis of the proteins are reviewed. Electrophoretic separation of proteins, procedures of transfer to membranes, immunological and nonimmunological protein detection systems, and characterization of protein-nucleic acid and protein-protein interactions are described. Emphasis is on the sensitivity of the methods described and on possible variations that allow the individual steps of Western blotting to be adapted to specific questions.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Svensson, H. (1961) Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. The differential equation of solute concentrations at a steady state and its solution for simple cases.Acta Chem. Scand. 15, 325–341.

    Article  CAS  Google Scholar 

  3. Renart, J., Reiser, J., and Stark, G. R. (1979) 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.

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

  5. Panyim, S. and Chalkey, R. (1969) High resolution acrylamide gel electrophoresis of histones.Arch. Biochem. Biophys. 130, 337–346.

    Article  CAS  Google Scholar 

  6. Margolis, J. and Kenrick, K. G. (1968) Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient.Anal. Biochem. 25, 347–362.

    Article  CAS  Google Scholar 

  7. Bjerrum, O. J., Selmer, J. C., and Lihme, A. (1987) Native immunoblotting: transfer of membrane proteins in the presence of non-ionic detergents.Electrophoresis 8, 388–397.

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  9. Otter, T., King, S. M., and Witman, G. B. (1987) A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins.Anal. Biochem. 162, 370–377.

    Article  CAS  Google Scholar 

  10. Lissilour, S. and Godinot, C. (1990) Influence of SDS and methanol on protein electrotransfer to Immobilon P membranes in semidry blot systems.Biotechniques 9, 397,398.

    CAS  Google Scholar 

  11. Tovey, E. R. and Baldo, B. A. (1987) Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes.Electrophoresis 8, 384–387.

    Article  CAS  Google Scholar 

  12. Otto, M. and Snejdarkova, M. (1981) A simple and rapid method for the quantitative isolation of proteins from polyacrylamide gels.Anal. Biochem. 111, 111–114.

    Article  CAS  Google Scholar 

  13. Protein Blotting Protocols (1991) Millipore Corp., Bedford, MA.

  14. Technical Information (1993) Bio-Rad Laboratories, Hercules, CA.

  15. Reig, J. and Klein, D. C. (1988) Submicrogram quantities of unstained proteins are visualized on polyvinylidene difluoride membranes by transillumination.Electrophoresis 1, 59,60.

    CAS  Google Scholar 

  16. Moeremans, M., Daneels, G., and De Mey, J. (1985) Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes.Anal. Biochem. 145, 315–321.

    Article  CAS  Google Scholar 

  17. Egger, D. and Bienz, K. (1987) Colloidal gold staining and immunoprobing of proteins on the same nitrocellulose blot.Anal. Biochem. 166, 413–417.

    Article  CAS  Google Scholar 

  18. Egger, D. and Bienz, K. (1992) Colloidal gold staining and immunoprobing on the same western blot, inMethods in Molecular Biology, vol. 10: Immunochemical Protocols (Manson, M., ed.), Humana, Totowa, NJ, pp. 247–253.

    Chapter  Google Scholar 

  19. Johnson, D. A., Gautsch, J. W., Sportsman J. R., and Elder, J. H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose.Gene Anal. Technol. 1, 3–8.

    Article  CAS  Google Scholar 

  20. Pfister, T., Pasamontes, L., Troxler, M., Egger, D., and Bienz, K. (1992) Immunocytochemical localization of capsid-related particles in subcellular fractions of poliovirus-infected cells.Virology 188, 676–684.

    Article  CAS  Google Scholar 

  21. Poor, M. L., Santa, P. F., and Sittampalam, G. S. (1988) Visualization of multiple protein bands on the same nitrocellulose membrane by double immunoblotting.Anal. Biochem. 175, 191–195.

    Article  CAS  Google Scholar 

  22. Moeremans, M., Daneels, G., de Raeymaeker, M., De Wever, B., and De Mey, J. (1987) The use of colloidal metal particles in protein blotting.Electrophoresis 8, 403–409.

    Article  CAS  Google Scholar 

  23. Kopriwa, B. M. (1975) A comparison of various procedures for fine grain development in electron microscopic radioautography.Histochemistry 44, 201–224.

    Article  CAS  Google Scholar 

  24. Bienz, K., Egger, D., and Pasamontes, L. (1986) Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.J. Histochem. Cytochem. 34, 1337–1342.

    CAS  Google Scholar 

  25. Sternberger, L. A., Hardy, P. H., Cucilis, J. J., and Meyer, H. G. (1970) The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes.J. Histochem. Cytochem. 18, 315–333.

    CAS  Google Scholar 

  26. Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., and Litt, G. J. (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays.J. Immunol Meth. 125, 279–285.

    Article  CAS  Google Scholar 

  27. Bronstein, I., Edwards, B., and Voyta, J. C. (1989) 1,2-dioxetanes: novel chemiluminescent enzyme substrates. Applications to immunoassays.J. Chemolumin. Biolumin. 4, 99–111.

    Article  CAS  Google Scholar 

  28. Schaap, A. P., Akhavan, A., and Romano, L. J. (1989) Chemiluminescent substrates for alkaline phosphatase: application to ultrasensitive enzyme-linked immunoassays and DNA probes.Clin. Chem. 35, 1863–1864.

    CAS  Google Scholar 

  29. Jones, A. and Moeremans M. (1988) Colloidal gold for the detection of proteins on blots and immunoblots, inMethods in Molecular Biology, vol. 3: New Protein Techniques (Walker, J. M., ed.), Humana, Clifton, NJ, pp. 441–79.

    Google Scholar 

  30. Ono, T. and Tuan, R. S. (1990) Double staining of immunoblots using enzyme histochemistry and india ink.Anal. Biochem. 187, 324–327.

    Article  CAS  Google Scholar 

  31. Szewczyk, B. and Summers, D. F. (1992) Fluorescent protein staining on nitrocellulose with subsequent immunodetection of antigen, inMethods in Molecular Biology, vol. 10: Immunochemical Protocols (Manson, M., ed.), Humana, Totowa, NJ, pp. 261–266.

    Chapter  Google Scholar 

  32. Glenney, J. (1986) Antibody probing of western blots which have been stained with india ink.Anal. Biochem. 156, 315–319.

    Article  CAS  Google Scholar 

  33. Jackson, P. and Thompson, R. J. (1984) The immunodetection of brain proteins blotted onto nitrocellulose from fixed and stained two-dimensional polyacrylamide gels.Electrophoresis 5, 35–42.

    Article  CAS  Google Scholar 

  34. Harper, D. R., Liu, K.-M., and Kangro, H. O. (1986) The effect of staining on the immunoreactivity of nitrocellulose bound proteins.Anal. Biochem. 157, 270–274.

    Article  CAS  Google Scholar 

  35. Tracy, R. P., Monkovic, D., Adrianorivo, A., Calore, J., and Mackay, A. (1987) The effect of prestaining prior to immunoreaction during electrophoretic blotting of proteins.Electrophoresis 8, 350–355.

    Article  CAS  Google Scholar 

  36. Bonner, W. M. and Laskey, R. A. (1974) A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels.Eur. J. Biochem. 46, 83–88.

    Article  CAS  Google Scholar 

  37. Laskey, R. A. and Mills, A. D. (1975) Quantitative film detection of3H and14C in polyacrylamide gels by fluorography.Eur. J. Biochem. 56, 335–341.

    Article  CAS  Google Scholar 

  38. Hawkes, R. (1982) Identification of concanavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting.Anal. Biochem. 123, 143–146.

    Article  CAS  Google Scholar 

  39. Schenkel, J., Sekeris, C. E., Alonso, A., and Bautz, E. K. F. (1988) RNA-binding properties of hnRNP proteins.Eur. J. Biochem. 171, 565–569.

    Article  CAS  Google Scholar 

  40. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting.Nucleic Acid Res. 8, 1–20.

    Article  CAS  Google Scholar 

  41. Fujimura, T. and Wickner, R. B. (1988) Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain.Cell 55, 663–671.

    Article  CAS  Google Scholar 

  42. Brimacombe, R., Stiege, W., Kyriatsoulis, A., and Maly, P. (1988) Intra- RNA and RNA-protein cross-linking techniques in E. coli ribosomes, inMethods in Enzymology, vol. 164, Academic Press, San Diego, CA, pp. 287–309.

    Google Scholar 

  43. Demczuk, S., Harbers, M., and Vennström, B. (1993) Identification and analysis of cell components of a gel retardation assay by combination with immunoblotting.Proc. Natl. Acad. Sci. USA 90, 2574–2578.

    Article  CAS  Google Scholar 

  44. Pellé, R. and Murphy, N. B. (1993) In vivo UV-cross-linking hybridization: a powerful technique for isolating RNA binding proteins. Application to trypanosome mini-exon derived RNA.Nucleic Acid Res. 21, 2453–2458.

    Article  Google Scholar 

  45. Carr, D. W. and Scott, J. D. (1992) Blotting and band-shifting. Techniques for studying protein-protein interactions.TIBS 17, 246–249.

    CAS  Google Scholar 

  46. Kaufmann, S. H., Ewing, C. M., and Sharper, J. H. (1987) The erasable Western blot.Anal. Biochem. 161, 81–95.

    Article  Google Scholar 

  47. Diano, M., Le Bivic, A., and Hirn, M. (1992) Raising polyclonal antibodies using nitrocellulose-bound antigen, inMethods in Molecular Biology, vol. 10: Immunochemical Protocols (Manson, M., ed.), Humana, Totowa, NJ, pp. 13–22.

    Chapter  Google Scholar 

  48. Szewczyk, B. and Summers, D. F. (1988) Preparative elution of proteins blotted to immobilon membranes.Anal. Biochem. 168, 48–53.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Medical Microbiology, University of Basel, Petersplatz 10, CH-4003, Basel, Switzerland

    Denise Egger & Kurt Bienz

Authors
  1. Denise Egger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kurt Bienz
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Egger, D., Bienz, K. Protein (western) blotting. Mol Biotechnol 1, 289–305 (1994). https://doi.org/10.1007/BF02921696

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02921696

Index Entries

Search

Navigation