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Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions
Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amena...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008431/ https://www.ncbi.nlm.nih.gov/pubmed/24788298 http://dx.doi.org/10.1371/journal.pone.0094251 |
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author | Petry, Franck R. Pelletier, Jérôme Bretteville, Alexis Morin, Françoise Calon, Frédéric Hébert, Sébastien S. Whittington, Robert A. Planel, Emmanuel |
author_facet | Petry, Franck R. Pelletier, Jérôme Bretteville, Alexis Morin, Françoise Calon, Frédéric Hébert, Sébastien S. Whittington, Robert A. Planel, Emmanuel |
author_sort | Petry, Franck R. |
collection | PubMed |
description | Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments. |
format | Online Article Text |
id | pubmed-4008431 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-40084312014-05-09 Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions Petry, Franck R. Pelletier, Jérôme Bretteville, Alexis Morin, Françoise Calon, Frédéric Hébert, Sébastien S. Whittington, Robert A. Planel, Emmanuel PLoS One Research Article Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments. Public Library of Science 2014-05-02 /pmc/articles/PMC4008431/ /pubmed/24788298 http://dx.doi.org/10.1371/journal.pone.0094251 Text en © 2014 Petry 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 Petry, Franck R. Pelletier, Jérôme Bretteville, Alexis Morin, Françoise Calon, Frédéric Hébert, Sébastien S. Whittington, Robert A. Planel, Emmanuel Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title | Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title_full | Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title_fullStr | Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title_full_unstemmed | Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title_short | Specificity of Anti-Tau Antibodies when Analyzing Mice Models of Alzheimer's Disease: Problems and Solutions |
title_sort | specificity of anti-tau antibodies when analyzing mice models of alzheimer's disease: problems and solutions |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008431/ https://www.ncbi.nlm.nih.gov/pubmed/24788298 http://dx.doi.org/10.1371/journal.pone.0094251 |
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