This article describes a modified yeast one-hybrid assay to detect interactions between heteromeric protein complexes and target DNA. The method involves using transformed yeast cells that express specific proteins and measuring β-galactosidase activity as an indicator of protein-DNA interactions.
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.
Resuspend the culture, and transfer 125 microliters from each well to a spectrophotometer plate. Measure the optical density at 600 nanometers to ensure it is between 0.3 and 0.6. Centrifuge the remaining cells in the deep well block at 3,000 times g and 21 degrees Celsius for 10 minutes. Remove the supernatant by inverting.
Add 200 microliters of Z-buffer to each well and vortex. Centrifuge at 3,000 times g and 21 degrees Celsius for 5 minutes. Remove the supernatant by inverting. Add 20 microliters of Z-buffer to each well and vortex. Cover the plate with sealing foil that is resistant to freeze-thaw cycles.
In a fume hood, perform four cycles of freeze-thaw using liquid nitrogen in a 42-degree Celsius water bath. Add 200 microliters of freshly-prepared Z-buffer/beta-mercaptoethanol/ONPG solution to each well. Incubate at 30 degrees Celsius for 17 to 24 hours or until color develops. Check that the color has developed.
Add 110 microliters of 1 molar sodium carbonate to each well to stop the reaction. Record the time, and vortex the plate. Centrifuge at 3,000 times g and 21 degrees Celsius for 10 minutes. Use a multichannel pipette to transfer 125 microliters of supernatant from each well to a spectrophotometer plate. Measure the optical density at 420 nanometers.
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