Antibody Validation by Western Blotting

Methods in Molecular Biology
Michele SignoreAlex Hodge

Abstract

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of W...Continue Reading

Citations

Dec 11, 2019·The Journal of Biological Chemistry·Lakshmi Pillai-KastooriAmy R Schutz-Geschwender
Jul 14, 2020·Frontiers in Neuroscience·Helena F FilippiniBarry J Sessle
Dec 20, 2020·Electrophoresis·Aristeidis NtoukasKonstantinos Poulas
Aug 22, 2021·Biotechnology Advances·Hèctor López-LagunaUgutz Unzueta
Oct 9, 2021·Journal for Immunotherapy of Cancer·Elisa BaldelliMariaelena Pierobon

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