ArticleYeast (Chichester, England)2009
Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.
Article in Yeast (Chichester, England), 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 68 papers.
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68 citing papers in PubMed.
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- CROP: a retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system.The EMBO journal · 2022Article
- What makes a histone variant a variant: Changing H2A to become H2A.Z.PLoS genetics · 2021Article
- Structure and activation mechanism of the hexameric plasma membrane HNature communications · 2021Article
- Translational activators and mitoribosomal isoforms cooperate to mediate mRNA-specific translation in Schizosaccharomyces pombe mitochondria.Nucleic acids research · 2021Article
- Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.Genes & development · 2021Article
- Diphthamide promotes TOR signaling by increasing the translation of proteins in the TORC1 pathway.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
8 more citing papers are in PubMed but not listed here.
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2 authors.
Funding
Abstract
PCR-mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope-tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR-based C-terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 x FLAG, T7, His-tag, Strep-tag II, S-tag, Myc, HSV, VSV-G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six-glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one-step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).
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