title: Generation and Validation of MYTH Baits: iMYTH and tMYTH Variants.
abstract: Generation of baits for membrane yeast two-hybrid (MYTH) screening differs depending on the nature of the protein(s) being studied. When using native yeast proteins with cytoplasmic carboxyl termini, the integrated form of MYTH (iMYTH) is the method of choice. iMYTH involves endogenous carboxy-terminal tagging of the gene of interest within the yeast chromosome, leaving the gene under the control of its natural promoter. When studying proteins not native to yeast, or native yeast proteins with only cytoplasmic amino termini, traditional MYTH (tMYTH) must be used. In the tMYTH approach, amino- or carboxy-terminally tagged proteins are expressed ectopically from a plasmid. In this protocol, we describe the generation and validation of iMYTH and tMYTH baits. MYTH bait generation can typically be completed in ∼1-2 wk.
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada;
authors.forename: Jamie
authors.initials: J
authors.lastname: Snider
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts
chemical_list: D008565:Membrane Proteins