Encyclopedia of Experiments: Biological Techniques
A subscription to JoVE is required to view this content.
Transcript
To detect interactions between heteromeric protein complexes involving multiple proteins with target DNA using the modified yeast one-hybrid assay, take a multi-well plate containing desired concentration of an actively growing, transformed yeast cell culture.
These cells contain a specific DNA sequence upstream of the reporter β-galactosidase-encoding gene, driven by the GAL-4 promoter, while lacking the endogenous GAL-4 transcription factor protein. The cells express a target protein fused to the GAL-4 activation domain and another protein lacking the GAL-4 DNA-binding domain.
If these proteins interact, they could form a complex, enabling the target protein to bind to the upstream DNA sequence in the reporter gene's promoter region. The GAL-4 activation domain activates reporter β-galactosidase enzyme expression.
Centrifuge. Resuspend the cells in a suitable buffer. Freeze-thaw the cells to lyse them and release β-galactosidase. Add beta-mercaptoethanol and a β-galactosidase substrate-containing buffer.
Beta-mercaptoethanol stabilizes the β-galactosidase enzyme. β-galactosidase hydrolyzes the β-galactosidase substrate, forming a yellow chromogen.
Add sodium carbonate solution to denature β-galactosidase and stop the reaction. Centrifuge. Transfer the supernatant containing yellow chromogen to a multi-well plate. Using a plate reader, measure the solution's optical density at a suitable wavelength to calculate the β-galactosidase activity.
Higher β-galactosidase activity indicates positive interactions between the proteins and target DNA sequence, which leads to β-galactosidase expression.
