Services

Online Inquiry

Electrophoretic Mobility Shift Assay (EMSA) Service

Inquiry

Plant Protein-Nucleic Acid Interaction Analysis

CD BioSciences provides Electrophoretic Mobility Shift Assay (EMSA) services for the analysis of protein-DNA and protein-RNA interactions in plant research. The service is suitable for studies involving plant transcription factors, RNA-binding proteins, nuclear protein extracts, purified proteins, promoter elements, and defined DNA or RNA motifs.

Depending on the research objective, EMSA can be used to determine whether a protein forms a detectable complex with a specific nucleic acid sequence, compare relative binding under different experimental conditions, and assess binding specificity using competitor probes, mutant probes, or antibody-based super-shift assays.

Principle of EMSA

EMSA is a non-denaturing gel electrophoresis method used to detect protein-nucleic acid complex formation. A labeled DNA or RNA probe is incubated with a protein sample under defined binding conditions. If the protein binds to the probe, the resulting protein-probe complex usually migrates more slowly than the free probe during native polyacrylamide gel electrophoresis.

The appearance of a shifted band indicates the formation of a protein-nucleic acid complex. Additional controls, such as excess unlabeled competitor probe, mutant competitor probe, or a target-specific antibody, are used to evaluate whether the observed shift is sequence-specific and associated with the target protein.

Schematic principle of electrophoretic mobility shift assay for protein-nucleic acid interaction analysis

Figure 1. Principle of EMSA showing the mobility difference between a free nucleic acid probe and a protein-probe complex.

Service Scope

The EMSA service can include experimental design, probe design, protein sample preparation or quality assessment, binding reaction setup, native PAGE, imaging, and data analysis. The project design is adjusted according to the protein type, probe sequence, sample source, antibody availability, and expected validation goal.

DNA EMSA

Analysis of interactions between plant transcription factors, nuclear extracts, or purified proteins and specific DNA sequences, promoter elements, or cis-regulatory motifs.

RNA EMSA

Analysis of interactions between RNA-binding proteins and defined RNA sequences, transcript fragments, RNA motifs, or candidate regulatory regions.

Specificity Validation

Use of unlabeled wild-type competitor probes, mutant probes, and non-specific competitors to evaluate sequence-specific binding.

Protein Identity Confirmation

Super-shift EMSA can be performed when a suitable target-specific antibody is available and compatible with the binding complex.

EMSA Assay Types

Assay Type	Purpose	Typical Design
Validation EMSA	Determines whether a protein sample forms a detectable complex with a labeled DNA or RNA probe.	Labeled probe, protein sample, and free probe control.
Competitive EMSA	Evaluates whether the shifted band is related to sequence-specific binding.	Excess unlabeled wild-type probe, mutant probe, or non-specific competitor probe.
Super-shift EMSA	Assesses whether the shifted complex contains the target protein.	Binding reaction with target-specific antibody and appropriate controls.

Typical lane layout for validation EMSA competitive EMSA and super-shift EMSA

Figure 2. Example lane arrangement for validation EMSA, competitive EMSA, and super-shift EMSA.

Probe Design and Labeling

Probe design is an important part of EMSA. A probe should contain the candidate binding sequence while avoiding strong secondary structure, self-annealing, or unrelated binding motifs where possible. For competitive EMSA, unlabeled wild-type probes, mutant probes, and non-specific competitor probes can be designed according to the experimental question.

If a probe sequence has not been finalized, CD BioSciences can assist with probe design based on the target protein, candidate promoter region, RNA sequence, predicted binding motif, species information, or published literature.

Available Probe Types

DNA probes
RNA probes
Labeled probes
Unlabeled competitor probes
Mutant probes
Non-specific competitor probes

Labeling Options

Non-radioactive labeled probes for routine EMSA detection
Isotope-labeled probes when very high sensitivity is required
Unlabeled probes for competition experiments

Experimental Controls

Appropriate controls are required for reliable EMSA interpretation. Depending on the assay type, the following controls may be recommended:

Free probe control
Probe with protein sample
Excess unlabeled wild-type competitor probe
Mutant competitor probe
Non-specific competitor probe
Antibody-treated sample for super-shift EMSA
Positive or negative control protein, if available
Protein concentration gradient, when binding strength comparison is required

These controls help distinguish sequence-specific binding from non-specific probe retention, protein aggregation, excessive background signal, or probe-related artifacts.

Workflow

Figure 3. General workflow of a customized EMSA project, from sequence and sample review to imaging and report delivery.

Review of target protein, nucleic acid sequence, sample type, and research objective.

Probe design, labeling strategy, and experimental control planning.

Protein sample preparation or protein quality assessment.

Probe synthesis, labeling, and preparation of competitor probes if required.

Binding reaction setup and adjustment of reaction conditions.

Native PAGE electrophoresis, signal detection, imaging, and data organization.

Sample Requirements

General sample requirements are listed below. The final requirements may vary depending on the number of groups, probe type, labeling method, and assay design.

Sample or Information	Recommended Requirement
Plant cells	More than 10⁷ cells for protein extraction.
Plant tissue	More than 2 g fresh or frozen tissue for protein extraction.
Nuclear protein or purified protein	More than 50 µL per experimental group is recommended.
Protein concentration	More than 0.5 mg/mL is recommended.
Purified protein	Purity above 90% is preferred for purified transcription factors or recombinant proteins.
Probe-related information	Probe sequence, candidate binding sequence, promoter region, RNA sequence, predicted motif, or relevant literature.
Antibody for super-shift EMSA	Target-specific antibody information is required when super-shift EMSA is planned.

Deliverables

Probe design information
Probe synthesis and labeling information
Labeled probe and unlabeled competitor probe information, if applicable
EMSA experimental record
Original gel images
Processed result images, if required
Data interpretation
Standard experimental report

Applications

Plant transcription factor binding site validation
Promoter and cis-element analysis
Plant stress response pathway research
Hormone signaling studies
Plant development-related gene regulation
RNA-binding protein functional studies
Verification of predicted DNA or RNA binding motifs
Comparison of binding activity among samples or treatments

Related EMSA Services

Project Information Required for Inquiry

For project evaluation, please provide the target protein name and species, protein source, candidate DNA or RNA sequence, probe sequence if already designed, expected assay type, antibody information if super-shift EMSA is planned, number of samples or treatment groups, and relevant references or preliminary data.

Please contact us to discuss the experimental design and sample requirements for your EMSA project.

Reference

Daras, G., et al. (2019). Detection of RNA-protein interactions using a highly sensitive non-radioactive electrophoretic mobility shift assay. Electrophoresis. 40(9), 1365-1371.

For research use only, not for clinical use.