EMSA/Gel Shift Assays

The EMSA (electrophoretic mobility shift assay) is used to study protein:DNA complexes and interactions. Protein:DNA complexes migrate more slowly than unbound linear DNA on a non-denaturing gel, causing a "shift."

Also called "gel shift" or "gel retardation" assays, the EMSA can be used to analyze sequence-specific recognition of nucleic acids by proteins.

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Figure 1. EMSA/gel shift assay diagram. When a large molar excess of unlabeled competitor DNA is added, the mobility shift is greatly reduced. Fluorescently labeled IRDye 700 oligonucleotide probes are used for detection. IRDye 700 oligos are end-labeled on both strands.

Advantages of Near-Infrared Fluorescence

Near-infrared fluorescence EMSA offers a safe, sensitive alternative to radioactive EMSA techniques.

You can easily adapt traditional, radioactive EMSA protocols to non-hazardous near-infrared fluorescence EMSA detection. Use IRDye® end-labeled oligonucleotides and image with the Odyssey® CLx Infrared Imager or Odyssey Classic Infrared Imager. You can perform the assay and obtain results in less than 2 hours with near-infrared fluorescence, as compared to several hours or overnight with other methods.

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EMSAs using near-infrared fluorescence technology are used to study:

  • Regulation of transcription
  • DNA replication
  • DNA repair
  • RNA processing
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Figure 2. EMSA/gel shift assay. IRDye 700 EMSA performed with consensus oligos for 3 different transcription factor targets. Arrows indicate positions of mobility shift.

You can detect protein:DNA complexes in the wet gel, without gel drying or film exposure. If needed, you can image the gel in the cassette on the Odyssey scanner surface to evaluate if the gel has run long enough, and if not, place it back in the chamber to electrophorese further.

Ready-to-use labeled oligos are available for a variety of common consensus sequences. Other IRDye end-labeled oligonucleotides and custom oligos are available through Integrated DNA Technologies (IDT), TriLink BioTechnologies, or Metabion International AG.

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Figure 3. AP-1 EMSA using IRDye 700 AP-1 oligonucleotide duplex. Nuclear extracts of HeLa cells treated with serum, were used to visualize increased AP-1 binding after serum stimulation. Competition reactions contained 100-fold molar excess of unlabeled oligo duplex. Arrow indicates mobility shift of fluorescent oligos. Asterisk indicates reduction of mobility shift caused by an excess of unlabeled competitor DNA. Wet gel was imaged with Odyssey Classic Infrared Imager.

Comparison of Infrared Fluorescent EMSA and Other Methods

Compare EMSA detection methods and find out how you can increase safety and save time using infrared detection.

IRDye® Infrared Fluorescence Dyes Radioisotopes Biotin/Streptavidin (Such as Thermo Scientific LightShift)
Total Time: 1.5h Total Time: 4.5 – 24h Total Time: 4.5 – 5h
Easy access and disposal Regulatory restrictions, disposal hassles, and cost Contact manufacturer for details
Fluorescent oligos have extended stability Short half-life of labeled oligos Chemiluminescent signal is unstable
Non-hazardous Hazardous Contact manufacturer for details
Wet gel imaged without removing gel plates Gel drying and film/phosphor-screen exposure required Membrane transfer is required
Fast, convenient, direct detection Time-consuming, inconvenient detection Indirect detection method. Blocking, streptavidin incubation, and washes are required
Gel can be replaced and run longer, if needed Gel run time cannot be extended Gel run time cannot be extended
Results available in less than 2 hours Results typically not obtained until the next day Detection steps add several hours to protocol

Typical EMSA Workflow

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