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Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies
Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225355/ https://www.ncbi.nlm.nih.gov/pubmed/22140431 http://dx.doi.org/10.1371/journal.pone.0026499 |
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author | Jost, K. Laurence Rottach, Andrea Milden, Manuela Bertulat, Bianca Becker, Annette Wolf, Patricia Sandoval, Juan Petazzi, Paolo Huertas, Dori Esteller, Manel Kremmer, Elisabeth Leonhardt, Heinrich Cardoso, M. Cristina |
author_facet | Jost, K. Laurence Rottach, Andrea Milden, Manuela Bertulat, Bianca Becker, Annette Wolf, Patricia Sandoval, Juan Petazzi, Paolo Huertas, Dori Esteller, Manel Kremmer, Elisabeth Leonhardt, Heinrich Cardoso, M. Cristina |
author_sort | Jost, K. Laurence |
collection | PubMed |
description | Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro. |
format | Online Article Text |
id | pubmed-3225355 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32253552011-12-02 Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies Jost, K. Laurence Rottach, Andrea Milden, Manuela Bertulat, Bianca Becker, Annette Wolf, Patricia Sandoval, Juan Petazzi, Paolo Huertas, Dori Esteller, Manel Kremmer, Elisabeth Leonhardt, Heinrich Cardoso, M. Cristina PLoS One Research Article Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro. Public Library of Science 2011-11-28 /pmc/articles/PMC3225355/ /pubmed/22140431 http://dx.doi.org/10.1371/journal.pone.0026499 Text en Jost 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 Jost, K. Laurence Rottach, Andrea Milden, Manuela Bertulat, Bianca Becker, Annette Wolf, Patricia Sandoval, Juan Petazzi, Paolo Huertas, Dori Esteller, Manel Kremmer, Elisabeth Leonhardt, Heinrich Cardoso, M. Cristina Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title | Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title_full | Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title_fullStr | Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title_full_unstemmed | Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title_short | Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies |
title_sort | generation and characterization of rat and mouse monoclonal antibodies specific for mecp2 and their use in x-inactivation studies |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225355/ https://www.ncbi.nlm.nih.gov/pubmed/22140431 http://dx.doi.org/10.1371/journal.pone.0026499 |
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