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Generation and characterization of a rat monoclonal antibody specific for multiple red fluorescent proteins.

Rottach, Andrea and Kremmer, Elisabeth and Nowak, Danny and Leonhardt, Heinrich and Cardoso, M Cristina (2008):
Generation and characterization of a rat monoclonal antibody specific for multiple red fluorescent proteins.
In: Hybridoma, pp. 337-43, 27, (5), ISSN 1557-8348, [Online-Edition: http://www.cardoso-lab.org/publications/Rottach_2008b.pdf],
[Article]

Abstract

Abstract Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in multiple colors spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a red fluorescent protein (DsRed) was discovered in the coral Discosoma sp. that provided a better spectral separation from cellular autofluorescence and allowed multicolor tracking of fusion proteins. However, the obligate tetramerization of DsRed caused serious problems for its use in live-cell imaging. Subsequent mutageneses of the red progenitor have resulted in several monomeric red FPs (mRFP1, mCherry, mOrange, mPlum, etc). These improved red FPs are characterized by higher brightness and photostability, complete chromophore maturation, and promise a wide variety of features for biological imaging and multicolor labeling. Here we report the generation and characterization of the first rat monoclonal antibody (MAb) against multiple red FPs, designated as multi-red 5F8. We demonstrate that multi-red 5F8 is a MAb with high affinity and specificity against the DsRed derivatives and corresponding fusion proteins, and that it is suitable for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence assays. Applying our versatile antibody, one and the same red fluorescent protein tag can be used to perform not only microscopic studies, but also multiple biochemical assays.

Item Type: Article
Erschienen: 2008
Creators: Rottach, Andrea and Kremmer, Elisabeth and Nowak, Danny and Leonhardt, Heinrich and Cardoso, M Cristina
Title: Generation and characterization of a rat monoclonal antibody specific for multiple red fluorescent proteins.
Language: English
Abstract:

Abstract Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in multiple colors spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a red fluorescent protein (DsRed) was discovered in the coral Discosoma sp. that provided a better spectral separation from cellular autofluorescence and allowed multicolor tracking of fusion proteins. However, the obligate tetramerization of DsRed caused serious problems for its use in live-cell imaging. Subsequent mutageneses of the red progenitor have resulted in several monomeric red FPs (mRFP1, mCherry, mOrange, mPlum, etc). These improved red FPs are characterized by higher brightness and photostability, complete chromophore maturation, and promise a wide variety of features for biological imaging and multicolor labeling. Here we report the generation and characterization of the first rat monoclonal antibody (MAb) against multiple red FPs, designated as multi-red 5F8. We demonstrate that multi-red 5F8 is a MAb with high affinity and specificity against the DsRed derivatives and corresponding fusion proteins, and that it is suitable for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence assays. Applying our versatile antibody, one and the same red fluorescent protein tag can be used to perform not only microscopic studies, but also multiple biochemical assays.

Journal or Publication Title: Hybridoma
Volume: 27
Number: 5
Divisions: 10 Department of Biology > Cell Biology and Epigenetics
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10 Department of Biology
Date Deposited: 06 Mar 2010 16:02
Official URL: http://www.cardoso-lab.org/publications/Rottach_2008b.pdf
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