An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Summary

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Abstract

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2^G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2^G73E proteins) as a biological example.

Introduction

EMSAs are used to analyze interactions between DNA and proteins by using native PolyAcrylamide Gel Electrophoresis (PAGE) to resolve a mixture of a protein of interest and a labeled DNA probe containing potential target sites of the protein¹. A DNA probe bound with protein will migrate slower compared with a free DNA probe, and is therefore retarded in its migration through a polyacrylamide matrix. Radiolabeling of DNA by ³²P has been the predominant method for detection in EMSAs. Although the application of radiolabeling in biochemical research has been beneficial, methods of alternative DNA labeling with comparable sensitivity are being employe....

Protocol

NOTE: EMSAs using fluorescently labeled DNA probes or other forms of labeled DNA share the same protocols for protein or cell extract preparation, protein-DNA binding reaction, and PAGE gel preparation and running (Figure 1A). The key differences are DNA labeling procedures, post-run gel processing steps, and detection methods.

1. Gel Preparation

1. Prepare 5% native polyacrylamide gel containing 0.5x TBE (45 mM Tris-Borate, 1 mM EDTA) and 2.5% glycerol, using a mini protein gel system (8.3 cm width x 7.3 cm length x 0.75 mm thickness).
   1. To prepare 30 mL of gel solution for 4 gels, mix 5 mL of 30% Acrylamide/Bis (3....

Results

Orange G loading dye (6x: 0.12 g Orange G in 100 mL 30% Glycerol) can be added to the binding reaction prior to loading to visualize the progress of the electrophoresis. Other loading dyes including bromophenol blue will be detected during scanning and therefore interfere with image analysis (Figure 1B).

In some instances of EMSAs, especially if nuclear extract preparation is used, poly deoxyinosinic-deoxycytidy.......

Discussion

fEMSAs are an efficient tool to investigate protein-DNA interactions, and are an alternative to radioactive labeling of DNA. Fluorescent dyes such as infrared dyes are commercially available, and provide a safer and more environmentally friendly method compared to radioactive DNA labeling. EMSAs using infrared fluorescent dye-labeled oligonucleotides do not require postrun gel processing steps, and therefore save time and cost compared to other DNA labeling techniques. The time to detect bands using radioactive EMSAs can.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30% Acrylamide/Bis Solution, 37.5:1	Bio-Rad	1610158	Acrylamide is harmful and toxic.
6x-His Epitope Tag Antibody (HIS.H8)	ThermoFisher	MA1-21315
Anti-Flag M2 antibody	Sigma-Aldrich	F3165-.2MG
Bovine Serum Albumin (BSA) molecular biology grade	New England Biolabs	B9000S
5'IRDye700-labeled DNA Oligos	Integrated DNA Technologies	Custom DNA oligo	These are referred to as "5'Dye-labeled or infrared fluorescent dye-labeled DNA oligos" in the manuscript. The company will custom synthesize 5' IRDye labeled-DNA oligonucleotides. Requires minimum 100 μM scale synthesis and HPLC purification.
Klenow Fragment (3'-->5' exo-)	New England Biolabs	M0212S
LightShif Poly (dI-dC)	ThermoFisher	20148E
Mini-PROTEAN Vertical Electrophoresis Cell	Bio-Rad	1658000FC	This is referred to as a 'mini protein gel system' in the manuscript. Any electrophoretic system can be used as long as they are clear glass plates of less than 25 cm x 25 cm in size.
Odyssey CLx Infrared Imaging System	LI-COR Biotechnology	Odyssey CLx Infrared Imagng System	This is referred to as an 'advanced infrared imaging system' in the manuscript.
Odyssey Fc Imaging System	LI-COR Biotechnology	Odyssey Fc Dual-Mode Imaging System	This is referred as a 'near-infrared fluorescent imaging system primarily for Western blots' in the manuscript.
Image Studio software (version 4.0)	LI-COR Biotechnology	Image Studio software	This is referred to as a 'particular imaging software' in the manuscript.
Orange G	Sigma-Aldrich	O3756-25G
6x Orange loading dye			0.25% Orange G; 30% Glycerol
0.5x TBE			45 mM Tris-Borate; 1 mM EDTA
1x TE			10 mM Tris-HCl, pH 8.0; 1 mM EDTA
1x STE			100 mM NaCl; 10 mM Tris-HCl, pH 8.0; 1 mM EDTA
5x Binding Buffer			50 mM Tris-HCl, pH 7.5; 50 mM NaCl; 200 mM KCl; 5 mM MgCl2; 5 mM EDTA, pH 8.0; 5 mM DTT; 250 μg/mL BSA