Schneider, B. L., Seufert, W., Steiner, B., Yang, Q. H., Futcher, A. B. (October 1995) Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast, 11 (13). pp. 1265-74. ISSN 0749-503X (Print)
Abstract
Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+ phenotype. Finally, selection for Ura- cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence Base Sequence Blotting, Western Epitopes Fungal Proteins/ analysis/immunology Genetic Vectors Polymerase Chain Reaction Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Saccharomyces cerevisiae/ chemistry/genetics Transformation, Genetic |
| Subjects: | Investigative techniques and equipment > cloning > PCR Investigative techniques and equipment > assays > cloning > PCR bioinformatics > genomics and proteomics > design > protein design organism description > yeast > Saccharomyces |
| CSHL Authors: | |
| Communities: | CSHL labs > Futcher lab |
| Depositing User: | Jessica Koos |
| Date: | October 1995 |
| Date Deposited: | 31 Jul 2014 15:03 |
| Last Modified: | 31 Jul 2014 15:03 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30640 |
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