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ProQuest™ Two-Hybrid System with Gateway™ Technology (Invitrogen™)

The ProQuest™ Two-Hybrid System with Gateway® Technology is an in vivo yeast-based system for identifying interactions between two proteins. This interaction reconstitutes a functional transcription factor that activates chromosomally integrated reporter genes where transcriptional activation is monitored by the growth of cells on selective media.
The ProQuest™ System features:
• Gateway® Technology to allow rapid and easy generation of bait and prey constructs, and to facilitate downstream application
• Low copy-number DBD (DNA Binding Domain) and AD (Activation Domain) vectors (ARS/CEN) to control over-expression and increase reproducibility
• Three different reporter genes (HIS3, URA3, and lacZ) with independent promoter regions to rapidly weed out false positives. URA3 reporter gene allows for both positive and negative selection, enabling advanced two-hybrid techniques such as reverse two-hybrid.

• A set of strong-to-weak Gateway®-based control interactions to evaluate results and assess the strength of the interaction being tested

Tango™ Open Enabling Model

The Tango™ Assay technology combines the benefits of the Tango™ assay platform with the highly accurate, sensitive, and live cell GeneBLAzer® Beta-lactamase reporter system. The Tango™ assay measures protein-protein interactions in live mammalian cells. One of the proteins of interest is anchored to cell surface either by its own transmembrane domains or through fusion to CD8 using a "bait" vector provided in this kit. The "bait" vector also provides at its intracellular C-terminus, an in-frame fusion to an exogenous transcription factor, GAL4-VP16. Interposed between the protein of interest and the transcription factor is a specific cleavage sequence for a non-native protease. This chimeric receptor protein is expressed in a cell line containing the beta-lactamase reporter gene responsive to the transcription factor. The "prey" vectors allow an interacting protein to be expressed as a protease fusion protein that recognizes and cleaves the site between the anchored membrane protein and transcription factor. The assay is performed by adding a stimulus to the growing cells for a defined period and measuring the activity of the reporter gene. Activation of the reporter gene provides a quantifiable measurement of the degree of interaction between the anchored membrane protein and the protease-tagged interaction partner and is unaffected by other signaling pathways in the cell, providing an exquisitely specific readout of protein-protein interaction.