Electrophoretic Mobility Shift Assay (EMSA) Service

  • Service Details
  • Case Study

Introduction of Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA), also known as gel retardation assay, is an important experimental method for studying the interaction of nucleic acids and proteins, and is the core technology for qualitative and quantitative analysis of interaction systems. This technology is often used to verify the interaction between transcription factors and promoters, and is now further developed to study DNA-protein interactions, RNA-protein interactions, and even DNA-RNA interactions.

EMSA is based on the principle that the mobility of nucleic acid-protein complexes is significantly reduced in native-PAGE, resulting in a lagged band in the final detection. In this experiment, specific and non-specific probes need to be designed so that they can bind to the target protein after incubation. As the molecular mass of the probe-protein complex increases, the immigration rates of the complex will reduce in SDS-PAGE compared with the probe that is not bound to the protein. Because there are some special markers in the probe (such as 32P), it can show bands by transmembrane, coloration or exposure to the probe-protein complex to prove the interaction between nucleic acid and protein. The experiment can also evaluate the binding affinity and even calculate the association and dissociation constant.

The schematic illustration of electrophoretic mobility shift assays (EMSA)Figure 1. The schematic illustration of electrophoretic mobility shift assays (EMSA) (Song, C.C.; et al. 2015)

Electrophoretic Mobility Shift Assay in Creative Proteomics

Creative Proteomics has accumulated nearly two decades of EMSA experience. Under the management of our professional technicians, a mature and high-quality EMSA platform has been established, which can help customers comprehensively and systematically analyze nucleotide-protein interactions. We provide a variety of radioactive isotopes and other labeling agents (such as biotin and FAM) to label nucleotides. Unlabeled nucleotides are used as specific competitors to eliminate non-specific nucleotide-protein interactions. In addition, our experts can customize experiments according to different project requirements, including but not limited to making changes in the following aspects:

  • Selection of nucleic acid target
  • Binding conditions
  • Additives
  • Competing nucleic acid
  • Electrophoresis conditions

Validating EMSA

Used to verify if the probe contains binding sites for the protein. Mutate the binding site, use the mutated probe as a control, and if wild-type probe shows a migration band while the mutated probe does not, it indicates the probe contains binding sites for the protein.

Competitive EMSA

The probe sequence remains unchanged, and the probe without modifying groups is called a cold probe. The cold probe competitively binds to the protein with the labeled probe. The cold probe does not reveal migration bands. Using the cold probe as a control, if the labeled probe shows migration bands and the cold probe migration bands weaken or disappear, false positives are eliminated, increasing experimental accuracy.

Super-Shift EMSA

Utilizes specific antibodies against the target protein. The protein-probe complex is recognized and bound by the antibody, causing a further increase in migration rate, resulting in a super-shift band above the protein-probe complex. This confirms the specific binding of the probe to the target protein.

Customers can choose different technology platforms according to project requirements, or contact us directly for consultation, and our expert team will provide you with customized experimental procedures.

Advantages of EMSA Services

  • Direct determination of protein-nucleic acid interactions, providing qualitative and quantitative information.
  • Can be used to study many types of interactions, such as specific binding, non-specific binding, sequence specificity, etc.
  • The technology is simple and the experiments are relatively easy to perform.
  • Different labeling methods and detection techniques can be used to adapt to different experimental needs.

Sample Preparation for EMSA

  • Cells >107, not less than 400 mg of animal tissues and not less than 2 g of plant tissues from which nucleoproteins are to be extracted
  • Probe sequence
  • If binding protein-specific antibody is used, the antibody is more than 50 μL and the concentration is more than 0.5 mg/mL

Applications of EMSA

EMSA is a widely used experimental technique for studying the interactions between proteins and nucleic acids (DNA or RNA). It finds broad applications in various research areas, including but not limited to the following:

Transcription Factor Studies: EMSA is employed to investigate the binding of transcription factors to DNA. Analyzing the binding capacity of transcription factors to specific gene promoters or regulatory elements reveals insights into gene regulatory networks and transcriptional control mechanisms.

RNA-Binding Protein Research: EMSA is used to study the interaction between RNA-binding proteins (RBPs) and RNA. Exploring the binding of RBPs to target RNA sequences provides understanding of RBPs' functions in processes such as post-transcriptional regulation, RNA stability, and translation control.

DNA Repair and Damage Recognition: EMSA is applied to study the binding of DNA repair enzymes and damage recognition proteins to DNA damage sites. Analyzing these interactions contributes to a deeper understanding of DNA repair mechanisms and DNA damage response pathways.

Drug Screening and Target Research: EMSA is utilized for screening and assessing the binding ability of potential drug molecules to specific DNA or RNA sequences. By detecting the interaction between drugs and targets, it evaluates drug affinity and selectivity, providing crucial information for drug development and targeted therapies.

Gene Regulatory Network Studies: EMSA is employed to investigate the interactions between transcription factors and regulatory elements in gene regulatory networks. Analyzing the binding of transcription factors to specific promoter or enhancer sequences reveals the structure and function of gene regulatory networks, as well as the role of transcription factors in regulating gene expression.

Functional Studies of Disease-Related Genes: EMSA is used to study the functional regulation of genes associated with diseases. Analyzing the binding of disease-related genes' promoters, enhancers, or regulatory elements to transcription factors provides insights into the regulatory mechanisms of these genes and functional changes associated with diseases.

Creative Proteomics is an international biotechnology company dedicated to research in molecular interactions and other related fields. The electrophoretic mobility shift assay we provided has the characteristics of high quality and efficiency, and the data obtained can be directly used for paper publication. Our one-stop service aims to save customers time and money.



  1. Hellman, L.M.; Fried, M.G. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interaction. Nat Protoc. 2007.
  2. Song, C.C.; et al. Choosing a suitable method for the identification of replication origins in microbial genomes. Front. Microbiol. 2015.

Case: Electrophoretic Mobility Shift Assay for Sensitive and Specific Detection of RNA and DNA Molecules


The traditional fluorometry method for detecting molecular beacons (MBs) has limitations, including sensitivity issues due to noise from free fluorophores and incomplete quenching. This study explores an alternative approach using gel electrophoresis for MB detection, providing an orthogonal confirmation of binding events and overcoming fluorometry drawbacks.


The study utilizes purified RNA samples obtained from red blood cells (RBCs) of healthy donors. Additionally, synthetic miRNA and DNA oligonucleotide target analogs are employed for testing the MB hybridization detection technique.

Technical Methods

MB Hybridization Detection by Gel Electrophoresis:

  • MBs (miR451aMB, miR486-5pMB, miR92a-3pMB, miR16-5pMB) incubated with various concentrations of synthetic miRNAs or DNA oligonucleotide target analogs.
  • Gel electrophoresis performed using Novex TBE 4–20% gels with constant voltage.
  • MB fluorescence signal visualized using ChemiDoc MP Imaging System.
  • Kinetic assay conducted at different time points (15 s to 30 min) with simultaneous sample loading.

Blood Draw and RBC Isolation:

  • Approval obtained from the Beth Israel Deaconess Medical Center Institutional Review Board.
  • Whole blood drawn from healthy volunteers using Vacutainer EDTA tubes.
  • RBCs isolated by dilution, filtration, and washing steps.

RNA Isolation, cDNA Synthesis, and qPCR:

  • RBC small RNA purified using miRNeasy Mini Kit.
  • RNA quantification performed with Qubit™ microRNA Assay Kit.
  • Gel electrophoresis TIFF images analyzed using ImageJ software.
  • Statistical analysis conducted for kinetic assay and dose-dependent assays.


1. Limitations of Fluorometry-Based MB Hybridization Readout:

  • Four MBs designed to detect specific miRNAs in RBCs and plasma.
  • In silico melting curve analysis and fluorescence measurements at different temperatures.
  • Background fluorescence impact on detection sensitivity discussed.

2. Hybridization of MB to the Target Alters Electrophoretic Properties:

  • Gel electrophoresis used as an alternative detection method to reduce MB fluorescence background.
  • Sensitivity tested with varying concentrations of miRNA target analogs.
  • Specific electrophoretic mobility patterns observed for different MBs.

3. Electrophoretic Mobility Can Identify Hybridization of MB to Mutated miRNA Target Analogs:

  • Ability of electrophoretic mobility shift to differentiate between wild type and mutated miRNA target sequences tested.
  • Comparison with fluorometry and flow cytometry for mutation detection.

4. Electrophoretic Mobility Shift Can Identify Endogenous miRNA Species:

  • Validation of the approach using purified RNA from RBCs.
  • Positive bands confirmed by miRNA inhibitors.
  • Gel electrophoresis applied to detect endogenous miRNA species in RBCs.

Electrophoretic mobility patterns of unbound and bound molecular beacons (MB).Electrophoretic mobility patterns of unbound and bound molecular beacons (MB).

Electrophoretic mobility shift differentiates certain hsa-miR-451a mutated sequences.Electrophoretic mobility shift differentiates certain hsa-miR-451a mutated sequences.


  1. Oliveira-Jr, Getulio P., et al. "Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection." Biosensors and Bioelectronics 189 (2021): 113307.
* This service is for RESEARCH USE ONLY, not intended for any clinical use.