Western Blotting: Immunoblotting

Abstract

Western blotting is a method by which proteins that have been physically separated and subsequently immobilized on the surface of a membrane are probed for reactivity with different types of affinity reagents, such as antibodies, receptors, are tested for the presence of interacting proteins in pure preparations or complex mixtures. Immunoblotting and the major steps of the procedure are described. It concerns the most commonly applied separation technique: sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE); the major transfer techniques: semidry or tank blotting; the many protein stains where the choice is depending on the sensitivity wanted and selection of the right blocking procedure. Visualization of bound probes represents a major issue as the variations are legio, but where the use of enzyme conjugated antibodies for colorimetric, chemo‐ and bioluminiscence is the most applied ones. The limits for quantitative use of the technique, the specificity problem crucial for a proper interpretation and various application, are described.

Key concepts

  • The nomenclature including geographical terms.

  • Overview of the many variants of the steps of the immunoblotting procedure.

  • Knowledge of the commonly appearing pitfalls of the technical process.

  • The limits for using immunoblotting for quantification purpose.

  • Understanding the importance of antibody specificity and use of controls for correct interpretation of the patterns.

Keywords: blotting; immunoblotting; western blotting; electroblotting; protein blotting

Figure 1.

General principle of electroimmunoblotting. Protein bands in a gel are electrotransferred to a membrane and subsequently probed with primary antibodies. After washing, the bound primary antibodies are detected by means of enzyme‐conjugated secondary antibodies that cause the development of precipitating colour after addition of the appropriate substrate. Modified from Bjerrum OJ and Heegaard NHH (eds) (1988) Handbook of Immunoblotting of Proteins, vol. I, p. 3. Copyright CRC Press, Boca Raton, FL, 1988.

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Bjerrum, Ole J, and Heegaard, Niels HH(Mar 2009) Western Blotting: Immunoblotting. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000995.pub2]