Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions

1. Day 9 (morning): Start the culture for enzymatic measurement.

1. Re-suspend culture and transfer 125 μL using a multichannel pipette on a spectrophotometer plate (measure OD₆₀₀).
   NOTE: Caution: Do not dispense the last drop to avoid bubbles, if the OD₆₀₀ is below 0.3 – 0.6, incubate the remaining cells for 1 – 2 h more based on the reading. The 125 μL culture used at this step can be discarded or transferred back to the original deep well block at the user's discretion.
2. Centrifuge the remaining cells in the deep well block for 10 min at 3000 x g and 21 °C. Remove supernatant by inverting.
3. Add 200 μL of Z-buffer and vortex.
   NOTE: Please see the recipe for Z-buffer in Table 1.
4. Centrifuge for 5 min at 3000 x g and 21 °C. Remove supernatant by inverting.
5. Add 20 μL Z-buffer and vortex.
6. Cover the plate with sealing foil that can resist freeze-thaw cycles. Perform 4 cycles of freeze/thaw using liquid nitrogen in a fume hood and then 42 ºC in a water bath (2 min each).
7. Add 200 μL Z-buffer/beta-mercaptoethanol (β-ME)/Ortho-nitrophenyl-β-galactoside (ONPG) (12 mL, 21 μL, 8.4 mg respectively will yield 20 mL solution; always prepare fresh).
   NOTE: ONPG will take some time to dissolve properly so prepare the solution an hour before reaching this step. ONPG serves as the substrate for the measurement of β-galactosidase activity.
8. Incubate for up to 17 – 24 h at 30 °C (check in between, if color develops earlier proceed to the next step).

2. Day 10 (morning): Stop the reaction and measure.

1. Add 110 μL 1M Na₂CO₃ to stop the reaction and record the time.
2. Vortex and centrifuge for 10 min at 3000 x g and 21 °C.
3. Take 125 μL of supernatant using multichannel and measure OD₄₂₀.
   NOTE: Caution, do not dispense the last drop to avoid bubbles.

Table 1:  The recipe for solutions used in the protocol. The table provides the recipe for the stock and working solutions of the major buffers used in the assay.