title: Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.
abstract: Before the advent of molecular methods to tag proteins, visualization of proteins within cells required the use of antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Epitope tagging allows the detection of all proteins with existing sequence information, irrespective of the availability of antibodies directed against them. This technique involves the generation of DNA constructs that express the protein of interest tagged with an epitope that can be recognized by a commercially available antibody. Proteins can be tagged with a wide variety of epitopes using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into mammalian cell lines and, in most cases, tightly mimic the behavior of the endogenous protein. Tagged proteins exogenously expressed in cells provide different types of information depending on the subsequent detection approaches. Using immunofluorescence and immunoelectron microscopy with anti-tag antibodies, relative to known markers of cellular organelles, can provide information on the subcellular localization of the tagged protein and may provide clues regarding the protein's function. Immunofluorescence with anti-tag antibodies can also be utilized to assess the tagged protein's responses to cellular signals and pharmacological treatments. Immunoprecipitations with anti-tag antibodies can recover protein complexes containing the protein of interest, resulting in the identification of interacting proteins. Recovery of tagged proteins on affinity matrices allows their purification for use in biochemical assays. In addition, specialized fluorescent tags, such as the green fluorescent protein (GFP) allow the analysis of cellular dynamics in live cells in real time.
authors: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA.
authors: Jay M
authors: JM
authors: Bhatt
authors: Department of Biology, Birmingham-Southern College, Birmingham, AL, USA.
authors: Melanie L
authors: ML
authors: Styers
authors: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA. esztul@uab.edu.
authors: Elizabeth
authors: E
authors: Sztul
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013194:Staining and Labeling / Q000379:methods*; D014162:Transfection
chemical_list: D000906:Antibodies; D000939:Epitopes; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors
keywords: Epitope tag; Green fluorescent protein (GFP); Immunofluorescence microscopy; Immunoprecipitation; Transfection; Western blot