Our yeast two-hybrid screening service includes:
  1. Bait self-activation test - This test determines whether baits activate transcription and whether the self-activation can be neutralized by inhibitors. This assay allows the determination of appropriate two-hybrid screening conditions for self-activating baits. Unlike other two-hybrid systems, our TetR-based systems work well with both self-activating and non-activating baits.

  2. Large-scale library screening - About 10 million independent cDNA library clones (about 5 x library coverage) are screened and all potential interactors are selected on the basis of their ability to grow on medium lacking uracil.

  3. Phenotypic analysis - Potential interactors are identified by examining the expression of two reporter genes under the control of TetOp in two independent basal promoters.

  4. False positives assay - Here we determine whether expression of the two-hybrid reporters is directly dependent on the interaction of TetOp with the bait fusion. The unique and powerful assay, which is available only with our TetR-based two-hybrid systems, allows the identification and elimination of false positives commonly associated with other two-hybrid systems.

  5. Validation - All potential interactors are isolated, then re-transformed to yeast strains that express the original bait or unrelated baits to further validate the interactions.
 

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