2017
DOI: 10.1007/978-1-4939-6857-2_10
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Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis

Abstract: We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is… Show more

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Cited by 11 publications
(7 citation statements)
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“…This selective enrichment may present an alternative method for removal of humics and other coextracting suppressants by isolating only proteins of interest, even low abundance ones, while leaving the remainder of the extract behind. Immunoprecipitation methodology could also increase confidence in the use of antibodies for direct analysis of fossil soft tissues because it can remove the concerns of nonspecific binding by validating the antibody target . Because mass spectrometry provides direct identification of isolated/enriched proteins, it is possible to directly evaluate the specificity of the antibodies/aptamers and rule out cross-reactivity with humics or other diagenetic molecules as the source of immunological response .…”
Section: Future Directions In Dtppmentioning
confidence: 99%
“…This selective enrichment may present an alternative method for removal of humics and other coextracting suppressants by isolating only proteins of interest, even low abundance ones, while leaving the remainder of the extract behind. Immunoprecipitation methodology could also increase confidence in the use of antibodies for direct analysis of fossil soft tissues because it can remove the concerns of nonspecific binding by validating the antibody target . Because mass spectrometry provides direct identification of isolated/enriched proteins, it is possible to directly evaluate the specificity of the antibodies/aptamers and rule out cross-reactivity with humics or other diagenetic molecules as the source of immunological response .…”
Section: Future Directions In Dtppmentioning
confidence: 99%
“…Once an antibody has been raised against a target protein (see Protocol: Raising Antibodies for Use in Xenopus [Piccinni and Guille 2020]), various strategies can be used to verify that the new antibody recognizes its target. Sometimes using immunoprecipitation, tryptic digest, and mass spectroscopy of the target protein is the only convincing approach (Persson et al 2017). In a well-established model like Xenopus, however, inducing loss or gain of function of the target protein in embryos followed by western blotting is a straightforward, powerful alternative strategy.…”
Section: Discussionmentioning
confidence: 99%
“…Antibodies are frequently incorporated in flow cytometry, characterizing patterns of a few proteins across tens of thousands of cells (34). However, antibodies present two primary hurdles for more comprehensive protein coverage: specificity and scalability (35)(36)(37)(38). Some new methods are attempting to increase protein multiplexing while maintaining or improving antibody specificity for protein quantitation in single cells, Figure 1.…”
Section: Antibody-based Methodsmentioning
confidence: 99%