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Antibody validation for Western blot: By the user, for the user

Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental...

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Autores principales: Pillai-Kastoori, Lakshmi, Heaton, Sam, Shiflett, Steve D., Roberts, Annabelle C., Solache, Alejandra, Schutz-Geschwender, Amy R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983856/
https://www.ncbi.nlm.nih.gov/pubmed/31819006
http://dx.doi.org/10.1074/jbc.RA119.010472
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author Pillai-Kastoori, Lakshmi
Heaton, Sam
Shiflett, Steve D.
Roberts, Annabelle C.
Solache, Alejandra
Schutz-Geschwender, Amy R.
author_facet Pillai-Kastoori, Lakshmi
Heaton, Sam
Shiflett, Steve D.
Roberts, Annabelle C.
Solache, Alejandra
Schutz-Geschwender, Amy R.
author_sort Pillai-Kastoori, Lakshmi
collection PubMed
description Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.
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spelling pubmed-69838562020-01-28 Antibody validation for Western blot: By the user, for the user Pillai-Kastoori, Lakshmi Heaton, Sam Shiflett, Steve D. Roberts, Annabelle C. Solache, Alejandra Schutz-Geschwender, Amy R. J Biol Chem Methods and Resources Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community. American Society for Biochemistry and Molecular Biology 2020-01-24 2019-12-09 /pmc/articles/PMC6983856/ /pubmed/31819006 http://dx.doi.org/10.1074/jbc.RA119.010472 Text en © 2020 Pillai-Kastoori et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Methods and Resources
Pillai-Kastoori, Lakshmi
Heaton, Sam
Shiflett, Steve D.
Roberts, Annabelle C.
Solache, Alejandra
Schutz-Geschwender, Amy R.
Antibody validation for Western blot: By the user, for the user
title Antibody validation for Western blot: By the user, for the user
title_full Antibody validation for Western blot: By the user, for the user
title_fullStr Antibody validation for Western blot: By the user, for the user
title_full_unstemmed Antibody validation for Western blot: By the user, for the user
title_short Antibody validation for Western blot: By the user, for the user
title_sort antibody validation for western blot: by the user, for the user
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983856/
https://www.ncbi.nlm.nih.gov/pubmed/31819006
http://dx.doi.org/10.1074/jbc.RA119.010472
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