Electrophoretic Mobility Shift Assay (EMSA) -

What is Electrophoretic Mobility Shift Assay (EMSA)?

Electrophoretic Mobility Shift Assay (EMSA) is an experimental method for study DNA binding proteins and RNA binding proteins. It was first applied in the field of biochemistry and molecular biology in the 1970s. Besides, EMSA technology separates DNA or RNA protein complexes and free nucleic acid molecules through electrophoresis to identify the binding ability of proteins to nucleic acids and to study their specificity and affinity.

The basic principle of EMSA technology is to form a complex between nucleic acid (DNA or RNA) and the protein to be tested, and then to electrophorese the complex and free nucleic acid molecules together in a gel electrophoresis chamber. The formation, size, and shape of the complex can be determined based on its electrophoretic mobility and position.

The steps of EMSA experiment include sample preparation and purification, preparation of electrophoresis buffer, loading amount of the sample, and electrophoresis time. In sample preparation, attention should be paid to the quality and purity of the sample, as well as the ratio and concentration of protein and nucleic acid. When setting the electrophoresis conditions, you should consider factors such as pH, ionic strength, and temperature for their impact on the experimental results.

The schematic illustration of electrophoretic mobility shift assays (EMSA) (Song et al., 2015).

Application of Electrophoretic Mobility Shift Assay

EMSA technology has a wide range of applications in biochemistry and molecular biology research, e.g. in gene regulation, transcription factors, DNA repair and cell signaling.

In gene regulation, we use EMSA to study the binding and function of transcription factors, as well as the effects of DNA methylation. In transcription factor research, we use EMSA technology to study the mechanisms of gene expression regulation, which provides insight into how transcription factors bind to DNA and regulate gene expression.

Electrophoretic mobility shift assay (EMSA) showing the association of SlHY5 with promoters of SGA biosynthetic genes SlGAME1, SlGAME4, and SlGAME17. (Wang et al., 2018)

In DNA repair, EMSA can help researchers understand the mechanisms by which DNA repair enzymes bind to DNA. For example, We can use EMSA technology to study the interaction between platinum drugs and the DNA-binding protein PARP-1 in the nucleus. And it can provide insight into how platinum drugs play a role in DNA damage repair.

In terms of cell signaling, the technique also allows analysis of protein-protein interactions in various cell signaling pathways. For instance, We also use EMSA technology to study the activity and localization of proteins in signaling pathways. Besides, this technique can help researchers better understand the complexity of cellular signaling pathways.

Advantages and Disadvantages of Electrophoretic Mobility Shift Assay

Advantages	Disadvantages
Enables direct measurement of affinity and specificity of DNA binding proteins	Sample preparation and experimental steps are cumbersome
Does not require large amounts of DNA samples	May not be sensitive to weakly bound proteins
Can determine the location and amount of DNA-binding proteins	Requires known DNA sequences to be used as probes
Can be used to screen for the effects of compounds or drugs on protein-DNA interactions	EMSA quantification results are influenced by many factors
Relatively fast, usually takes a few hours to obtain results	Cannot measure specific binding constants
Relatively low cost	Only suitable for studies where binding proteins are known

Comparison of EMSA

Techniques	Advantages	Disadvantages
EMSA	High sensitivity and specificity, can detect protein-DNA/RNA interactions in vitro, low sample consumption	Time-consuming, requires purified protein and labeled nucleic acid, can produce non-specific binding
DNA gel shift assay	Simple and easy to perform, requires minimal equipment, can detect DNA-protein interactions	Low sensitivity and specificity, can produce non-specific binding, cannot detect RNA-protein interactions
EMMA	High resolution, can detect complex protein-DNA interactions, can be used for in vivo studies	Time-consuming, requires purified protein and labeled nucleic acid, complex data analysis
Affinity chromatography	High purity of protein, can be used for large-scale purification, can be used for in vivo studies	Low sensitivity, requires prior knowledge of protein-ligand interaction, cannot detect protein-DNA/RNA interactions directly

Creative Proteomics is a leading provider of Electrophoretic Mobility Shift Assay (EMSA) services. And we can help you achieve the following studies:

Studies of RNA-binding proteins, such as transcription factor-related studies
Study of protein-protein interactions, etc.

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References

Song, Chengcheng, Shaocun Zhang, and He Huang. “Choosing a suitable method for the identification of replication origins in microbial genomes.” Frontiers in microbiology 6 (2015): 1049.
Wang, Cui-cui, et al. “Manipulation of light signal transduction factors as a means of modifying steroidal glycoalkaloids accumulation in tomato leaves.” Frontiers in Plant Science 9 (2018): 437.