title: 1D4: a versatile epitope tag for the purification and characterization of expressed membrane and soluble proteins.
abstract: Incorporation of short epitope tags into proteins for recognition by commercially available monoclonal or polyclonal antibodies has greatly facilitated the detection, characterization, localization, and purification of heterologously expressed proteins for structure-function studies. A number of tags have been developed, but many epitope-antibody combinations do not work effectively for all immunochemical techniques due to the nature of the tag and the specificity of the antibodies. A highly versatile, multipurpose epitope tag is the 9 amino acid C-terminal 1D4 peptide. This peptide tag together with the Rho1D4 monoclonal antibody can be used to detect proteins in complex mixtures by western blotting and ELISA assays, localize proteins in cells by immunofluorescence and immunoelectron microscopic labeling techniques, identify subunits and interacting proteins by co-immunoprecipitation, and purify functionally active proteins including membrane proteins by immunoaffinity chromatography. In this chapter we describe various immunochemical procedures which can be used for the detection, purification and localization of 1D4-tagged proteins for structure-function studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada, V6T 1Z3.
authors.forename: Laurie L
authors.initials: LL
authors.lastname: Molday
authors.forename: Robert S
authors.initials: RS
authors.lastname: Molday
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins