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A Detailed and Radioisotope-free Protocol for Electrophoretic Mobility Shift Assay (EMSA)
Published: Aug 20, 2020 DOI: 10.21769/BioProtoc.3721 Views: 3647
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Abstract
To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. In the present protocol, DNA probes are labeled with Biotin. Therefore, it is safer for researchers when they do not need to use radioisotope-labeled probes. In addition, this protocol is to provide a detailed procedure for a successful EMSA assay. The interested recombinant protein can be mixed with putative-targeted DNA probes for a binding reaction at room temperature (25 °C). Afterward, the reaction mixture can be run in a native polyacrylamide gel and transferred to a positively charged nylon membrane. Finally, the results can be detected and visualized via the biotin-streptavidin chemiluminescence.
Keywords: DNA-protein interaction
DNA binding protein
Electrophoretic mobility shift assay
EMSA
Transcription factor
Background
EMSA is a powerful method to detect the direct interaction between a protein (e.g., transcription factor) and a DNA fragment (Chen, 2011). Typically, a protein-bound DNA probe moves slower than the free-DNA probes in a gel. This protein-DNA complex can be observed as a shifted band and the free-DNA probes move faster toward the bottom of the gel. Based on this characteristic, the DNA probes can be labeled with a radioactive isotope [e.g., Phosphorus-32 (32P)] or Biotin and the shifted band can be visualized via autoradiography or chemiluminescence, respectively. Several previous EMSA protocols have been published (Hellman and Fried, 2007; Chen, 2011). In these protocols, the DNA probes were labeled with a radioisotope, 32P (Hellman and Fried, 2007; Chen, 2011). The use of radioisotope is not only dangerous for researchers but also requires specific laboratory infrastructure which is strictly controlled for safety. Here, a detailed and radioisotope-free protocol for EMSA assay is described. This protocol was successfully applied to test the binding of different transcription factors to their targeted DNA fragments in my lab (data not shown).
Materials and Reagents
- 1.5 ml micro-tubes (Eppendorf, catalog number: 00 30121589 )
- Positively charged nylon membrane (GE Healthcare AmershamTM HybondTM-N+) (Thermo Fisher Scientific, catalog number: 45-000-927 )
- Interested recombinant proteins
- Labeled and unlabeled (3′-end Biotin) oligo DNA probes (sense and anti-sense)
Note: In this protocol, the length of oligos is 40 bp. The biotin is only added to 3′-end of sense oligo. These oligos can be ordered from a local company and it also provides a service which can label oligo with biotin. If there is no such that service, the PierceTM Biotin 3' End DNA Labeling Kit (Thermo Fisher Scientific, catalog number: 89818 ) can be considered. In addition, it is better to include one more EMSA assay (for negative control) with a non-targeted DNA probe. - Tris pH 7.5 (1 M) (Thermo Fisher Scientific, catalog number: 15567027 )
- NaCl (Sigma-Aldrich, catalog number: S7653 )
- KCl (Sigma-Aldrich, catalog number: P9333 )
- DTT (Dithiothreitol) (Sigma-Aldrich, catalog number: 3483-12-3)
- Glycerol (Sigma-Aldrich, catalog number: G5516 )
- MgCl2 (Sigma-Aldrich, catalog number: M8266 )
- Poly(dI.dC) (Thermo Fisher Scientific, catalog number: 20148E )
Note: The synthetic polymer poly(dI.dC) which can function as a non-specific competitor is added to reduce the nonspecific interaction. - NP-40 (Thermo Fisher Scientific, catalog number: 28324 )
- 30% Acrylamide/Bis Solution, 29:1 (Bio-Rad, catalog number: 1610156 )
- TEMED (N,N,N',N'-tetramethylethylenediamine) (Bio-Rad, catalog number: 161-0801 )
- APS (ammonium persulfate) (Sigma-Aldrich, catalog number: A3678 )
- 6x DNA Gel Loading Dye (Thermo Fisher Scientific, catalog number: R0611 )
- Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, catalog number: 89880 )
- Tris base
- Boric acid
- EDTA
- 1 M and 100 mM DTT (see Recipes)
- 10x TBE buffer (see Recipes)
Equipment
- Centrifuge (e.g., Eppendorf, model: 5418R )
- Heat-block (e.g., Eppendorf, model: Eppendorf ThermoMixer® C)
- -20 °C freezer
- Vertical electrophoresis apparatus (e.g., Mini-PROTEAN® system, Bio-Rad, US)
- Gel transferring apparatus (e.g., Mini Trans-Blot® system, Bio-Rad, US)
- UV-cross-linker (e.g., Stratalinker® UV Crosslinker)
- Dancer orbital shaker (e.g., Phoenix, model: RS-OS 10/20 )
- Bio-imaging system equipped with a charge-coupled device (CCD)-camera (e.g., ChemiDoc, Bio-Rad, US)
Procedure
Category
Molecular Biology > DNA > DNA-protein interaction
Molecular Biology > DNA > Electrophoresis
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