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Antibody Validation by Western Blotting

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Molecular Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1606))

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 Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

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Acknowledgment

This work was supported in part by George Mason University and the Istituto Superiore di Sanità, Rome, Italy.

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Authors and Affiliations

  1. Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Via Giano della Bella 34, 00162, Rome, Italy

    Michele Signore Ph.D.

  2. Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy

    Valeria Manganelli

  3. George Mason University, Center for Applied Proteomics and Molecular Medicine, Manassas, VA, USA

    Alex Hodge

Authors
  1. Michele Signore Ph.D.
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  2. Valeria Manganelli
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  3. Alex Hodge
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Corresponding author

Correspondence to Michele Signore Ph.D. .

Editor information

Editors and Affiliations

  1. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, USA

    Virginia Espina

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Signore, M., Manganelli, V., Hodge, A. (2017). Antibody Validation by Western Blotting. In: Espina, V. (eds) Molecular Profiling. Methods in Molecular Biology, vol 1606. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6990-6_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6990-6_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6989-0

  • Online ISBN: 978-1-4939-6990-6

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