TU Darmstadt / ULB / TUbiblio

Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies.

Jost, K. Laurence and Rottach, Andrea and Milden, Manuela and Bertulat, Bianca and Becker, Annette and Wolf, Patricia and Sandoval, Juan and Petazzi, Paolo and Huertas, Dori and Esteller, Manel and Kremmer, Elisabeth and Leonhardt, Heinrich and Cardoso, M. Cristina (2011):
Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies.
In: PloS one, pp. e26499, 6, (11), ISSN 1932-6203,
[Article]

Abstract

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.

Item Type: Article
Erschienen: 2011
Creators: Jost, K. Laurence and Rottach, Andrea and Milden, Manuela and Bertulat, Bianca and Becker, Annette and Wolf, Patricia and Sandoval, Juan and Petazzi, Paolo and Huertas, Dori and Esteller, Manel and Kremmer, Elisabeth and Leonhardt, Heinrich and Cardoso, M. Cristina
Title: Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies.
Language: English
Abstract:

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.

Journal or Publication Title: PloS one
Volume: 6
Number: 11
Divisions: 10 Department of Biology > Cell Biology and Epigenetics
?? fb10_zoologie ??
10 Department of Biology
Date Deposited: 13 Dec 2011 09:10
Export:
Suche nach Titel in: TUfind oder in Google

Optionen (nur für Redakteure)

View Item View Item