Overview
In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.
Protocol
1. Day 9 (morning): Start the culture for enzymatic measurement.
- 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. - Centrifuge the remaining cells in the deep well block for 10 min at 3000 x g and 21 °C. Remove supernatant by inverting.
- Add 200 μL of Z-buffer and vortex.
NOTE: Please see the recipe for Z-buffer in Table 1. - Centrifuge for 5 min at 3000 x g and 21 °C. Remove supernatant by inverting.
- Add 20 μL Z-buffer and vortex.
- 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).
- 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. - 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.
- Add 110 μL 1M Na2CO3 to stop the reaction and record the time.
- Vortex and centrifuge for 10 min at 3000 x g and 21 °C.
- 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.
| TE buffer 10X (10ml) | |
| Desirable | From the stock |
| 1000mM Tris-Cl (pH 8.0) | 1mL of Tris 1M |
| 10mM EDTA (pH 8.0) | 200uL of EDTA 0.5M |
| Make up the volume with water | |
| TE/LiAc (10ml) | |
| Desirable | From the stock |
| 1X | 1mL TE 10X |
| 0.1M LiAc | 1mL LiAc 1M |
| Make up the volume with water | |
| TE/LiAc/PEG (50 mL) 50% | |
| Desirable | From the stock |
| 1X | 5 mL TE 10X |
| 0.1M LiAc | 5 mL LiAc 1M |
| 40 mL PEG3350 | |
| Z-buffer (1L) pH 7.0 | |
| Na2HPO4 16.1g of heptahydrated or 8.52g of anhydrous | |
| NaH2PO4 5.5g of hydrated or 4g of anhydrous | |
| KCl 0.75g | |
| MgSO4 0.246g of hydrated or 0.12g of anhydrous | |
| Maintain pH with HCl | |
| All solutions are sterlized before use. | |
| Media and agar plates used in the protocol (YPDA, SD-Ura; SD-Trp-Ura and SD-Trp-Ura agar plates) follow the same recipe as in Y1H | |
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Materials
| Name | Company | Catalog Number | Comments |
| 96-well round bottom Plate | Greiner bio-one | 650101 | Plastic ware |
| 96-deep well block | USA Scientific | 1896-2000 | Plastic ware |
| Sealable Foil | USA Scientific | 2923-0110 | Plastic ware |
| Araseal | Excel Scientific | B-100 | Plastic ware |
| 2-mercaptoethanol | Fisher Scientific | 034461-100 | Assay |
| ONPG | Sigma-Aldrich | 73660 | Assay |
| Na2CO3 | Sigma-Aldrich | 223484 | Assay |
| Na2HPO4 | Sigma-Aldrich | S3264 | Assay |
| NaH2PO4 | Sigma-Aldrich | S3139 | Assay |
| KCl | Fisher Scientific | BP366-500 | Assay |
| MgSO4 | Sigma-Aldrich | 83266 | Assay |
| HCl | Fisher Scientific | SA54-4 | Assay |
| Drybath | Thermo Fischer | Device | |
| Vortex | Thermo Fischer | Device | |
| Centrifuge | Eppendorf | Centrifuge 5810R | Device |
| Plate Reader | Molecular Device | SPECTRAMAX PLUS Microplate Spectrophotometer | Device |
| Incubator | Thermo Fisher | Model No. 5250- 37 Degree, 6250-30 degrees | Device |
| Shaker Incubator | New Brunswick | Device | |
| Water Bath | Thermo Fisher | IsoTemp 205 | Device |
| Tris Base | Fisher Scientific | BP152-1 | Assay |
