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

Overview

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

1. 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 different modifications to the binding buffer.
2. Right before setting up binding reactions, pre-run the 5% native polyacrylamide gel in 0.5x TBE + 2.5% glycerol to remove all traces of ammonium persulfate at 80 V for 30 - 60 min, or until the current no longer varies with time.
3. Set up the binding reaction in the final volume of 20 µL.
   1. Mix 4 µL of 5x binding buffer, 80 - 200 ng purified protein A (in 50% glycerol, i.e., SOX-2), 1 µL of 0.1 µM Dye-conjugated probe (final concentration: 5 nM), and ddH₂O.
   2. Optional: When cold probe competitors are required, add various concentrations (2x, 10x, 25x, 50x, 100x, etc.) of cold probes.
   3. Optional: When the interaction between proteins A and B (i.e., SOX-2 and LIM-4) is tested, add 80 - 200 ng purified protein B (in 50% glycerol, i.e., LIM-4).
   4. Optional: When nuclear extract preparation is used and the specific binding of protein A is to be validated, add an antibody specific to protein A (i.e., anti-6xHis, anti-FLAG, etc.).
   5. Incubate at R/T in the dark for 15 min.
4. Load all of the binding reactions onto the gel and run the gel at 10 V/cm to the desired distance.

2. Imaging

NOTE: The gel was scanned directly in the glass plates with an advanced infrared imaging system. Therefore, the gel can be resolved further and scanned repeatedly (Figures 1C and 2). A near-infrared fluorescent imaging system primarily for Western blots was also tested to scan the gel, but only the advanced infrared imaging system was able to scan the gel with good resolution. The methodology described is specific to a particular infrared imaging software, although other software packages may be used.

1. Clean the bed of the scanner with ddH₂O and dry well before scanning. Wipe dry the glass plates of the gel and place the plates containing the gel on the scanner bed.
2. Open the infrared imaging software and go to the 'Acquire' tab. When the thinner plate (1 mm) is placed on the scanner bed, use the settings of Channel: 700, Intensity: Auto, Resolution: 169 µm, Quality: medium, and Focus offset: 1.5 mm. When the thicker plate (3 mm) is placed on the scanner bed, use the settings of Channel: 700, Intensity: Auto, Resolution: 84 µm, Quality: medium, and Focus offset: 3.5 mm. Focus offset depends on the thickness of the glass plate.
3. Select the area that the gel occupies on the scanner. Click 'Start' to begin the scan.

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-2^G73E to the SOX-2 target site of the LIM-4/SOX-2 element. 5 nM of 5'Dye-LIM-4/SOX-2 probe was used. LIM-4/SOX-2(M), mutant target sites. The gels were scanned at 20 and 40 min after electrophoresis. The scanned image obtained at 40 min after electrophoresis was used to quantify the fluorescent band intensity, which was normalized by the intensity of the lane without any cold probe. AU, arbitrary unit.

Figure 2. Supershift Analysis of the SOX-2-DNA Complex using fEMSA and Antibodies. SOX-2 was tagged with both 6xHis and FLAG epitopes. Addition of anti-6xHis or anti-FLAG antibodies caused a supershift of the SOX-2-DNA complex. 5'Dye-LIM-4/SOX-2 probe was used. Gel images were spliced between lanes 2 and 3 to exclude irrelevant lanes. The gel was scanned at 40, 60, and 90 min after electrophoresis.

Materials

Name	Company	Catalog Number	Comments
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