Barbara Magi and Sabrina Liberatori

Summary

Immunoblotting techniques use antibodies (or other specific ligands in related techniques) to identify target proteins among a number of unrelated protein species. They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions. Proteins are typically separated by electrophoresis and transferred onto membranes (usually nitrocellulose). The membrane is overlaid with a primary antibody for a specific target and then with a secondary antibody labeled, for example, with enzymes or with radioisotopes. When the ligand is not an antibody, the reaction can be visualized using a ligand that is directly labeled. Dot blot is a simplified procedure in which protein samples are not separated by electrophoresis but are spotted directly onto membrane. Immunoblotting is now widely used in conjunction with two-dimensional polyacrylamide gel electrophoresis, not only for traditional goals, such as the immunoaffinity identification of proteins and analysis of immune responses but also as a genome-proteome interface technique.

Key Words: Western blotting; immunoblotting; electrophoresis; 2D electrophoresis; immunoaffinity identification; immunoblotting techniques.

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition.

Edited by: R. Burns © Humana Press Inc., Totowa, NJ

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