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

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.

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. Remo....

Materials

List of materials used in this article

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