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.
Similar content being viewed by others
References
Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (Lond.) 227, 680–685.
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.
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.
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.
Panyim, S. and Chalkey, R. (1969) High resolution acrylamide gel electrophoresis of histones.Arch. Biochem. Biophys. 130, 337–346.
Margolis, J. and Kenrick, K. G. (1968) Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient.Anal. Biochem. 25, 347–362.
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.
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.
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.
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.
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.
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.
Protein Blotting Protocols (1991) Millipore Corp., Bedford, MA.
Technical Information (1993) Bio-Rad Laboratories, Hercules, CA.
Reig, J. and Klein, D. C. (1988) Submicrogram quantities of unstained proteins are visualized on polyvinylidene difluoride membranes by transillumination.Electrophoresis 1, 59,60.
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.
Egger, D. and Bienz, K. (1987) Colloidal gold staining and immunoprobing of proteins on the same nitrocellulose blot.Anal. Biochem. 166, 413–417.
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.
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.
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.
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.
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.
Kopriwa, B. M. (1975) A comparison of various procedures for fine grain development in electron microscopic radioautography.Histochemistry 44, 201–224.
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.
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.
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.
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.
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.
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.
Ono, T. and Tuan, R. S. (1990) Double staining of immunoblots using enzyme histochemistry and india ink.Anal. Biochem. 187, 324–327.
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.
Glenney, J. (1986) Antibody probing of western blots which have been stained with india ink.Anal. Biochem. 156, 315–319.
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.
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.
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.
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.
Laskey, R. A. and Mills, A. D. (1975) Quantitative film detection of3H and14C in polyacrylamide gels by fluorography.Eur. J. Biochem. 56, 335–341.
Hawkes, R. (1982) Identification of concanavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting.Anal. Biochem. 123, 143–146.
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.
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.
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.
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.
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.
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.
Carr, D. W. and Scott, J. D. (1992) Blotting and band-shifting. Techniques for studying protein-protein interactions.TIBS 17, 246–249.
Kaufmann, S. H., Ewing, C. M., and Sharper, J. H. (1987) The erasable Western blot.Anal. Biochem. 161, 81–95.
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.
Szewczyk, B. and Summers, D. F. (1988) Preparative elution of proteins blotted to immobilon membranes.Anal. Biochem. 168, 48–53.
Author information
Authors and Affiliations
Rights 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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02921696