Yeast Two-Hybrid Systems Protocols

Directed Yeast Two Hybrid Screening for MEKK1 Interaction Partners Protocol Protocol utilizes the Y190 yeast strain, that characteristically turns from white to red during growth due to ademutation. Baits were subcloned into the pAS1cyh2 vector, and Preys subcloned into pACT2.

Two Hybrid System Transformation Protocol High Efficiency Transformation of a yeast strain requiring maintenance of a plasmid.

Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator.

Two-hybrid Systems Stage 2: Selecting an Interactor Protocol In this stage of the protocol, a mammalian cDNA library constructed in a plasmid such as pJG4-5 is transformed into yeast strains containing pBAIT and the lexAop-lacZ reporter plasmid. PJG4-5 expresses the cloned cDNAs from a cassette containing a transcriptional activation domain and other moieties under the control of the yeast GAL1 promoter.

Yeast Two-hybrid Systems Protocol Protocol for Yeast two-hybrid systems.