Cargando…

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investig...

Descripción completa

Detalles Bibliográficos
Autores principales: Gilda, Jennifer E., Ghosh, Rajeshwary, Cheah, Jenice X., West, Toni M., Bodine, Sue C., Gomes, Aldrin V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545415/
https://www.ncbi.nlm.nih.gov/pubmed/26287535
http://dx.doi.org/10.1371/journal.pone.0135392
_version_ 1782386750194188288
author Gilda, Jennifer E.
Ghosh, Rajeshwary
Cheah, Jenice X.
West, Toni M.
Bodine, Sue C.
Gomes, Aldrin V.
author_facet Gilda, Jennifer E.
Ghosh, Rajeshwary
Cheah, Jenice X.
West, Toni M.
Bodine, Sue C.
Gomes, Aldrin V.
author_sort Gilda, Jennifer E.
collection PubMed
description Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.
format Online
Article
Text
id pubmed-4545415
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-45454152015-09-01 Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS) Gilda, Jennifer E. Ghosh, Rajeshwary Cheah, Jenice X. West, Toni M. Bodine, Sue C. Gomes, Aldrin V. PLoS One Research Article Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis. Public Library of Science 2015-08-19 /pmc/articles/PMC4545415/ /pubmed/26287535 http://dx.doi.org/10.1371/journal.pone.0135392 Text en © 2015 Gilda et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Gilda, Jennifer E.
Ghosh, Rajeshwary
Cheah, Jenice X.
West, Toni M.
Bodine, Sue C.
Gomes, Aldrin V.
Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title_full Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title_fullStr Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title_full_unstemmed Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title_short Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)
title_sort western blotting inaccuracies with unverified antibodies: need for a western blotting minimal reporting standard (wbmrs)
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545415/
https://www.ncbi.nlm.nih.gov/pubmed/26287535
http://dx.doi.org/10.1371/journal.pone.0135392
work_keys_str_mv AT gildajennifere westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs
AT ghoshrajeshwary westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs
AT cheahjenicex westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs
AT westtonim westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs
AT bodinesuec westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs
AT gomesaldrinv westernblottinginaccuracieswithunverifiedantibodiesneedforawesternblottingminimalreportingstandardwbmrs