Written and maintained by CASRAI Editorial Board
Last updated
Short answer
An EMSA (electrophoretic mobility shift assay, also called a gel shift assay) detects protein-nucleic acid binding on a native gel. A protein-bound nucleic acid migrates more slowly than the same nucleic acid unbound, so the bound and free forms appear as separate bands. It shows that binding happens, not which protein is responsible or where on the nucleic acid it sits.
Definition and scope
The method was reviewed in detail by Hellman and Fried in Nature Protocols (2007), which describes protein-nucleic acid complexes as migrating more slowly than the corresponding free nucleic acid on a native gel. Thermo Fisher Scientific’s Pierce protein methods page on gel shift assays says the same of protein-DNA complexes relative to free linear DNA fragments. The gel is run under native (non-denaturing) conditions so that complexes survive electrophoresis, which is the main difference from SDS-PAGE, where proteins are denatured. For the general principle of separating molecules in a gel, see what gel electrophoresis is.
How it is used in practice
The nucleic acid probe carries a label so that the free and bound forms can be seen. The probe is mixed with a purified protein or a crude extract, the mixture is resolved on a native gel, and the positions of the bands are compared with a probe-only lane. The readout is the appearance of a slower band and the loss of free probe as more protein is added.
Two practical points shape how results are read:
- Crude extracts contain many proteins. Hellman and Fried note that non-specific competitors such as genomic DNA, poly d(A-T) or poly d(I-C) reduce binding by secondary proteins, but they also reduce specific binding, so the amount of competitor needs to be titrated rather than fixed.
- The reaction is not at equilibrium during the run. Hellman and Fried state that samples are not at chemical equilibrium during the electrophoresis step, so a complex that dissociates quickly can be missed or under-represented.
Because EMSA is a gel-based binding readout, it is often discussed alongside other interaction methods such as co-immunoprecipitation, which asks a different question (which proteins associate) rather than whether a protein binds a given nucleic acid sequence.
Label and detection options
The probe label determines sensitivity and workflow.
| Label or detection | What the Hellman and Fried and Thermo Fisher pages say |
|---|---|
| 32P end-labelling | End-labelling the nucleic acid with 32P-phosphate is described by Hellman and Fried as a widely used approach. Thermo Fisher notes DNA probes have traditionally been radiolabelled with 32P. |
| Biotin or digoxigenin (hapten) labels | Detected with streptavidin or anti-DIG after the gel is transferred to a membrane (Thermo Fisher). Hellman and Fried describe biotin labels as detected by chromophore deposition. |
| Fluorescent probes | Imaged in the gel. Thermo Fisher says this has not been very popular because expensive instruments are required; Hellman and Fried describe fluorophore labelling as less sensitive than radioisotope labelling. |
| Unlabelled probe, stained afterwards | Hellman and Fried describe using unlabelled nucleic acid and detecting it by staining after electrophoresis. Thermo Fisher notes that this detection method requires a lot of DNA, and carrier and competitor DNA can also stain and raise background. |
Control checklist: what makes a shift interpretable
A slower band alone is weak evidence. Hellman and Fried and Thermo Fisher describe the controls that turn it into a defensible result:
- Non-specific competitor. Sonicated salmon sperm DNA or poly(dI.dC) (Thermo Fisher), or genomic DNA, poly d(A-T) or poly d(I-C) (Hellman and Fried), titrated because it can also lower specific binding.
- Specific competitor. Unlabelled copies of the probe. Thermo Fisher states that, generally, a 200-fold molar excess of unlabelled probe is sufficient to out-compete specific interactions.
- Antibody supershift. An antibody against a candidate protein slows the complex further, which supports that protein’s presence. Thermo Fisher adds two points: the disappearance of a shifted band with no supershift is a negative result, and an antibody can disrupt the protein-DNA interaction, losing the shift without producing a supershift.
Hellman and Fried also describe two-dimensional approaches that pair EMSA with Western blotting or mass spectrometry to identify the protein in a shifted band; see Western blot basics for the immunodetection side.
What a shift does not tell you
- Identity. A shift does not reveal which protein is bound, or its molecular weight. Supershift or a 2D approach is needed.
- Binding site. A shift does not show where on the nucleic acid the protein binds. Hellman and Fried point to footprinting for that question.
- Conditions outside the method’s range. Hellman and Fried limit EMSA samples to 300 mM salt or less (1:1 salt) and to DNAs of up to about 5000 bp.
- Fast kinetics. Hellman and Fried note that the roughly one minute needed to mix the reaction components and for the sample to migrate into the gel limits kinetic studies of rapid binding.
Where it matters for labs
For a lab deciding whether EMSA fits a question, a simple split follows from the methods above. Use it to test whether a given protein or extract binds a given sequence and to compare binding under different conditions or with competitors. Choose a different method when the question is the binding site (footprinting), the identity of an unknown binder (supershift, Western blot or mass spectrometry follow-up) or fast kinetics. The label chosen also shapes the workflow: hapten labels add a membrane transfer and detection step, while fluorescent probes are imaged in the gel, which Thermo Fisher notes needs expensive instruments. Related nucleic acid detection formats are covered in the Southern blot guide, which probes DNA fixed on a membrane rather than in a native binding reaction.
Common misunderstandings
- “A shifted band proves a specific interaction.” Not without a specific competitor, a non-specific competitor and, ideally, a supershift or a matching control.
- “More competitor is always cleaner.” Competitor can lower specific binding as well as background, so it is titrated.
- “No shift means no binding.” The gel is not at equilibrium, so a rapidly dissociating complex may not survive the run.
- “A supershift is required for every result.” It is a way to identify a candidate protein; an antibody can also disrupt the complex without a supershift appearing.
Frequently asked questions
What does EMSA stand for?
Electrophoretic mobility shift assay. It is also called a gel shift assay.
What does an EMSA detect?
The binding of a protein to a nucleic acid, seen as a slower-migrating complex on a native gel compared with the free nucleic acid.
How do you confirm which protein caused the shift?
By adding an antibody to a candidate protein (supershift), or by pairing EMSA with Western blotting or mass spectrometry as described by Hellman and Fried.
Sources
- Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols 2007;2(8):1849-1861. https://pmc.ncbi.nlm.nih.gov/articles/PMC2757439/ (checked 2026-10-29).
- Thermo Fisher Scientific, Pierce protein methods: gel shift assays (EMSA). https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html (checked 2026-10-29).
