EMSA (gel shift)
Show that a protein binds a DNA or RNA probe.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Holding a complex together long enough to see it
In an electrophoretic mobility shift assay a short labelled DNA or RNA probe is mixed with protein, the mixture is loaded on a non-denaturing gel, and an electric field pulls everything towards the anode. Free probe is small and highly charged and runs quickly.
2From a shifted band to an affinity and a specificity
A titration with the probe far below KD and the protein over a wide range gives the fraction of probe shifted in each lane; the protein concentration at half-shift is then close to KD, and a binding equation fitted to all lanes gives a value with a confidence range. The gel is not at equilibrium, so the number reflects what survived the run as well as what was in the tube, and is best treated as an estimate.
Free probe
Fast band near the bottom; it should be sharp and alone in the no-protein lane.
Complex
Slower band whose share of the lane signal rises with protein.
Competition
Unlabelled specific DNA removes the shift; mutant-site DNA does not.
Supershift
An antibody that slows or blocks the complex identifies the protein in it.
3How it goes wrong
DNA-binding proteins also bind any DNA weakly, so without an excess of unrelated carrier such as poly(dI·dC) extra molecules load onto the probe at high protein: slower ladders and smears appear, signal collects in the well, and the specific band fades. Too much carrier, on the other hand, can compete with weak specific binding.
Key terms
Practise these as flashcards →A short labelled DNA or RNA containing the binding site, used at a concentration well below KD for affinity measurements.
The slower band of probe held in a protein complex.
Stabilisation of a complex inside the gel matrix, where a dissociated protein is likely to rebind before escaping.
Unrelated DNA such as poly(dI·dC) added in excess to absorb weak, sequence-independent binding.
Unlabelled probe with the binding site; it removes the shifted band if the binding is specific.
Unlabelled DNA with the site mutated; it should leave the shifted band intact.
A further reduction in mobility when an antibody binds the protein in the complex, identifying it.
Running the native gel before loading to remove persulfate and establish an even ion distribution.
When to use it
- Showing that a protein binds a particular DNA or RNA sequence, and which nucleotides matter, using wild-type and mutant probes and competitors.
- Estimating the affinity of a purified protein for its site, or comparing affinities across mutants or sites.
- Detecting a DNA-binding activity in nuclear or cell extracts and identifying the protein responsible by supershift.
- Resolving complexes of different stoichiometry or composition, for example one versus two proteins on a probe, which solution methods average together.
Compared with alternatives
Measures binding at true equilibrium in a plate and suits titrations and screens; it cannot resolve several complexes or show what is in them.
ChIP-based methods show where a protein binds in the genome in cells; prefer them for occupancy in vivo, and EMSA for direct binding to a defined sequence.
Show whether a site affects transcription in cells, which binding alone does not establish.
Maps protein–RNA interactions in cells; RNA EMSA tests direct binding to a defined RNA in vitro.