The yeast two-hybrid assay detects binary protein-protein interactions by separating a transcription factor into a DNA-binding domain fused to a "bait" protein and a transcriptional activation domain fused to a "prey" protein; if bait and prey interact in yeast, the transcription factor is reconstituted and activates reporter genes such as HIS3, ADE2, lacZ, MEL1, or other selectable/readable reporters. The readout is yeast growth on selective medium and/or reporter activity, which reflects proximity-dependent transcriptional activation in the yeast nucleus rather than direct biochemical binding in the original mammalian, tumor, neuronal, macrophage, or organoid context. Because yeast two-hybrid can generate false positives and false negatives, interaction claims should be validated using independent assays such as co-immunoprecipitation, Western blot, immunofluorescence colocalization, BiFC, pull-down, or mammalian two-hybrid assays.
Yeast Two-Hybrid Assay (Y2H)
Yeast Two-Hybrid (Y2H) is a molecular biology technique used to study protein interactions. This method uses the isolation of transcription factors in yeast cells to detect protein-protein interactions. In a sensing system, two complementary fragments of a target protein bind to parts that interact with two different proteins, triggering the reassembly of the transcription factor. This technique is useful for screening and validating the importance of protein-protein interactions in biology.
Related Experimental Schemes
