Abstract
We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of protein-DNA interactions in vivo by chromatin immunoprecipitation (ChIP). The system is based on coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a better signal/noise ratio.
MeSH terms
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Animals
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Biotinylation*
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Carbon-Nitrogen Ligases / chemistry*
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Carbon-Nitrogen Ligases / genetics
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Cell Cycle Proteins / genetics
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Chromatin / chemistry*
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Chromatin / genetics
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Chromatin / immunology
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Chromobox Protein Homolog 5
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Chromosomal Proteins, Non-Histone / genetics
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DNA-Binding Proteins / genetics
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Escherichia coli / genetics
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Green Fluorescent Proteins
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HeLa Cells
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Humans
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Immunosorbents
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Luminescent Proteins / genetics
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Mice
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NIH 3T3 Cells
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Precipitin Tests*
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Promoter Regions, Genetic / genetics
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Protein Binding / genetics
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Repressor Proteins / chemistry*
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Repressor Proteins / genetics
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Transcription Factor DP1
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Transcription Factors / chemistry*
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Transcription Factors / genetics
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Transduction, Genetic
Substances
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Cell Cycle Proteins
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Chromatin
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Chromosomal Proteins, Non-Histone
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DNA-Binding Proteins
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Escherichia coli Proteins
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Immunosorbents
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Luminescent Proteins
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Nuclear Proteins
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Recombinant Fusion Proteins
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Repressor Proteins
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Transcription Factor DP1
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Transcription Factors
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Chromobox Protein Homolog 5
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Green Fluorescent Proteins
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Carbon-Nitrogen Ligases
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birA protein, E coli