Yeast three-hybrid

Service Introduction

The yeast three-hybrid system was improved from the yeast two-hybrid system by introducing a plasmid pBridge that could simultaneously insert two exogenous proteins The bait protein was inserted into the MCS I of this plasmid, and the N terminus was still fused to the DNA-binding domain BD of the GAL4 system An exogenous protein regulated by the PMET25 promoter was inserted into the MCS II. The PMET25 promoter has the property of being able to start only whenionine is deficient, which makes it possible to regulate the expression of the exogenous protein downstream. Similarly, the pBridge plasmid was transformed into the AH109 strain, and then mated with the library working solution transformed into Y187 for screening positive clones.

Service Advantages

▶Upgraded plasmid system, precise carrying of two exogenous proteins;

▶Controllable PMET25 promoter, significantly reduced experimental interference;

▶Classic strain pairing, stable and efficient screening system;

▶Can be integrated with our other interaction experiments, allowing the entire experiment to be completed smoothly and quickly.

Service Process

Article Case

A plant NLR receptor employs ABA central regulator PP2C-SnRK2 to activate antiviral immunity

academic journal：nature communications

Yeast three-hybrid (Y3H) assays showing the effect of Sw-5b NB on the interaction between NbPP2C4 and NbSnRK2.3 or between NbPP2C4 and NbSnRK2.4. Yeast cells were cotransformed with pGAD-NbPP2C4 and pBridge-NbSnRK2.3/2.4 + YFP or pGADNbPP2C and pBridge-NbSnRK2.3/2.4 + Sw-5b NB domain. The interactions were assayed on SD/-TL (lacking Trp and Leu) and SD/-TLHAM (lacking Trp, Leu, His, Ade, and Met) dropout medium plates, respectively, for 3–5 days.

Applicable Fields

▶Verify protein interactions: Detect whether there are interactions among the three proteins;

▶Study protein complexes: Analyze the formation mechanisms and functions of multi-protein complexes;

▶Explore signaling pathways: Investigate the cooperative actions of multiple proteins in signaling pathways.