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Related Concept Videos

Electrophoresis: Overview01:20

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
Two-dimensional Gel Electrophoresis01:22

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as  cells...
Capillary Electrophoresis: Instrumentation01:20

Capillary Electrophoresis: Instrumentation

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components...
DNA Agarose Gel Electrophoresis02:35

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
Southern Blot02:57

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

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Related Experiment Video

Updated: Jun 14, 2026

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
09:58

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Published on: November 29, 2016

Principles and problems of the electrophoretic mobility shift assay.

Neil S Holden1, Claire E Tacon

  • 1Department of Cell Biology and Anatomy, Airway Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. nholden@ucalgary.ca

Journal of Pharmacological and Toxicological Methods
|March 30, 2010
PubMed
Summary

The electrophoretic mobility shift assay (EMSA) detects DNA binding proteins by observing slower migration of protein-bound DNA in gels. This study demonstrates EMSA

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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
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Last Updated: Jun 14, 2026

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
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Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
12:44

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Published on: August 21, 2016

Area of Science:

  • Molecular Biology
  • Biochemistry

Background:

  • The electrophoretic mobility shift assay (EMSA) is a standard technique for identifying DNA-binding proteins.
  • EMSA relies on the principle that protein-bound DNA exhibits reduced mobility in a polyacrylamide gel compared to free DNA.

Purpose of the Study:

  • To detail the classical electrophoretic mobility shift assay (EMSA) protocol.
  • To demonstrate the application of EMSA in identifying specific protein-DNA interactions.

Main Methods:

  • Nuclear protein isolation using sequential membrane lysis.
  • Radiolabeling of DNA probes with Phosphorus-32 ((32)P) using T4 polynucleotide kinase.
  • Incubation of proteins and probes in binding buffer, with optional antibody for supershift analysis, followed by non-denaturing polyacrylamide gel electrophoresis and autoradiography.

Main Results:

  • EMSA successfully detected induced DNA-protein complexes in stimulated A549 cells (TNFα and PMA) compared to unstimulated controls.
  • Supershift analysis confirmed the presence of p50 and p65 subunits of NF-κB within these complexes.

Conclusions:

  • The study provides a comprehensive protocol for EMSA, including troubleshooting.
  • Limitations, variants, and alternative techniques to EMSA are discussed.