Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions
Published: June 29, 2023
Abstract
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.
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 wel…
Divulgations
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citer Cet Article
Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions. J. Vis. Exp. (Pending Publication), e21436, doi: (2023).