Source: Tripathi, P. et al., A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies. J. Vis. Exp. (2017)
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.
1. Day 9 (morning): Start the culture for enzymatic measurement.
2. Day 10 (morning): Stop the reaction and measure.
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 |
The authors have nothing to disclose.
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 |