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.
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