title: Biochemical approaches including the design and use of strains expressing epitope-tagged proteins.
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.
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA.
authors.forename: Mary T
authors.initials: MT
authors.lastname: Couvillion
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D007158:Immunologic Techniques / Q000379:methods*; D048648:Macronucleus / Q000737:chemistry / Q000235:genetics; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D030781:Organisms, Genetically Modified / Q000235:genetics; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016053:RNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000235:genetics; D019076:Transgenes
chemical_list: D000345:Affinity Labels; D016054:DNA, Protozoan; D000939:Epitopes; D046912:Multiprotein Complexes; D015800:Protozoan Proteins; D016053:RNA, Protozoan