title: Transient expression of epitope-tagged proteins in mammalian cells.
abstract: Before the advent of molecular methods to tag proteins, the visualization of proteins within cells by immunoelectron microscopy required the use of highly specific antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Current technologies allow the detection of proteins for which specific antibodies are not available. This procedure involves the generation of DNA constructs that express the protein of interest tagged with an epitope that is recognized by a well-characterized commercially available antibody. Proteins can be tagged with a wide variety of epitopes, small and large, using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into many mammalian cell lines and, in most cases, tightly mimic the distribution of the endogenous protein. Prior to immunoelectron microscopy, expression and localization of tagged proteins can be assessed by Western blotting and immunofluorescence. Furthermore, specialized fluorescent tags, such as the green fluorescent protein (GFP), can be used to rapidly screen for transfection efficiency and localization. The use of epitope-tagged protein expression has increased the versatility of immunoelectron microscopy to explore the function of uncharacterized proteins for which highly specific antibodies are not available.
authors.affiliation: Department of Biology, Chemistry, and Mathematics, University of Montevallo, Montevallo, AL, USA.
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Styers
authors.forename: Jason
authors.initials: J
authors.lastname: Lowery
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sztul
mesh_terms: D002478:Cells, Cultured; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology
chemical_list: D000939:Epitopes; D011993:Recombinant Fusion Proteins