Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes
Published: June 29, 2023
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…
Divulgazioni
The authors have nothing to disclose.
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 |
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Citazione di questo articolo
Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes. J. Vis. Exp. (Pending Publication), e21432, doi: (2023).