Abstract
Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit. Common biochemical methods for detecting protein, RNA, and DNA in Tetrahymena thermophila are also discussed.
Copyright © 2012 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Affinity Labels / chemistry*
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DNA, Protozoan / chemistry
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DNA, Protozoan / isolation & purification
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Epitope Mapping / methods*
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Epitopes / chemistry*
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Gene Expression Regulation
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Gene Transfer Techniques
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Genetic Loci
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Immunologic Techniques / methods*
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Macronucleus / chemistry
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Macronucleus / genetics
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / isolation & purification
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Organisms, Genetically Modified / genetics
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Promoter Regions, Genetic
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Protozoan Proteins / chemistry
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Protozoan Proteins / isolation & purification*
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RNA, Protozoan / chemistry
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RNA, Protozoan / isolation & purification
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Tetrahymena thermophila / chemistry*
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Tetrahymena thermophila / genetics
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Transgenes
Substances
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Affinity Labels
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DNA, Protozoan
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Epitopes
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Multiprotein Complexes
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Protozoan Proteins
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RNA, Protozoan