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

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Citer Cet Article
Modified Yeast-One Hybrid Assay to Detect Heteromeric Protein Complex-DNA Interactions. J. Vis. Exp. (Pending Publication), e21436, doi: (2023).

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