Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes

Abstract

Source: Hsieh, Y. W. et al., An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides. J. Vis. Exp. (2016)

In this video, we demonstrate the electrophoretic mobility shift assay to detect protein-DNA interactions using infrared fluorescent-labeled DNA probes. When the protein binds to the DNA probe, it forms a large complex that migrates slowly on and appears as a shifted band on the gel.

Protocol

1. Binding Reaction and Electrophoresis Prepare 1 mL of 5x binding buffer by mixing 50 µL of 1 M Tris-HCl, pH 7.5; 10 µL of 5 M NaCl; 200 µL of 1 M KCl, 5 µL of 1 M MgCl2, 10 µL of 0.5 M EDTA, pH 8.0; 5 µL of 1 M DTT; 25 µL of 10 mg/mL BSA, and 695 µL of ddH2O. NOTE: The 5x Binding Buffer can be aliquoted and stored at -20 °C. Another point to consider is that different transcription factors will have d…

Representative Results

Figure 1. fEMSA using Infrared Fluorescent Dye-labeled Oligonucleotides. (A) Flow chart of fEMSA. (B) The effect of bromophenol blue dye, orange G, and dI-dC (1 µg) on fEMSA. 5 nM of 5'Dye-LIM-4/SOX-2 probe was used.(C)fEMSA showing binding specificity of 6xHis-SOX-2G73E to the SOX-2 target site of the LIM-4/SOX-2 element. 5 nM of 5'Dye-LIM-4/SOX-2 probe w…

Materials

30% Acrylamide/Bis Solution, 37.5:1	Bio-Rad	1610158	Acrylamide is harmful and toxic.
Bovine Serum Albumin molecular-biology-grade	New England Biolabs	B9000S
5'IRDye700-labeled DNA Oligos	Integrated DNA Technologies	Custom DNA oligo	These are referred 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 100μM scale synthesis and HPLC purification.
Mini-PROTEAN Vertical Electrophoresis Cell	Bio-Rad	1658000FC	Device
Odyssey CLx Infrared Imaging System	LI-COR Biotechnology	Odyssey CLx Infrared Imagng System	Device
Odyssey Fc Imaging System	LI-COR Biotechnology	Odyssey Fc Dual-Mode Imaging System	Device
Image Studio software (version 4.0)	LI-COR Biotechnology	Image Studio software	Software
Orange G	Sigma-Aldrich	O3756-25G	Chemical
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; 1 mM EDTA, pH8.0
1x STE			100 mM NaCl; 10 mM Tris-HCl, pH8.0; 1 mM EDTA
5x Binding Buffer			50 mM Tris-HCl, pH7.5; 50 mM NaCl; 200 mM KCl; 5 mM MgCl2; 5 mM EDTA, pH8.0; 5 mM DTT; 250 ug/ml BSA