EMSA and DNA footprinting are classic methods for studying protein-DNA interactions. EMSA detects binding and measures affinity, while footprinting identifies the specific nucleotides that are contacted by the binding protein.
Electrophoretic Mobility Shift Assay
EMSA, also called gel shift assay, is based on the observation that protein-DNA complexes migrate more slowly through a native polyacrylamide gel than free DNA, similar to agarose gel electrophoresis. A radiolabeled or fluorescently labeled DNA probe containing the putative binding site is incubated with a protein extract or purified protein. The mixture is separated by native gel electrophoresis. Free DNA runs faster, while protein-bound DNA is shifted to a higher position in the gel.
EMSA is highly sensitive and can detect interactions in complex mixtures. Specificity is demonstrated by competition with unlabeled specific and non-specific DNA. Excess unlabeled specific DNA eliminates the shifted band, while non-specific competitor does not. Supershift assays use antibodies against the binding protein to further retard the complex, confirming the protein identity.
Binding Affinity and Stoichiometry
EMSA can measure binding affinity by titrating protein concentration and quantifying the fraction of bound DNA. The equilibrium dissociation constant is determined from the protein concentration at half-maximal binding. Multiple shifted bands can reveal different stoichiometric complexes or multiple binding sites. Cooperative binding is detected when the binding curve is sigmoidal rather than hyperbolic.
Probe Design
DNA probes for EMSA are typically 20 to 40 base pairs containing the binding site. Double-stranded probes are prepared by annealing complementary oligonucleotides and end-labeling with radioactive phosphorus-32 or fluorescent dyes. Longer probes of several hundred base pairs can be used for mapping cis-regulatory regions. Biotin-labeled probes with chemiluminescent detection, as used in Southern blot, provide a non-radioactive alternative.
DNase I Footprinting
DNase I footprinting identifies the specific nucleotides protected by a bound protein. A DNA fragment labeled at one end is incubated with the binding protein and then partially digested with DNase I. The nuclease cuts unprotected DNA stochastically, but the protein-bound region is protected from cleavage. The digestion products are separated by denaturing gel electrophoresis alongside a sequencing ladder.
The protection pattern appears as a gap in the ladder of bands, the footprint. The boundaries of the footprint define the protein-binding site at nucleotide resolution. Hypersensitive sites, where DNase I cuts more frequently, are often seen at the edges of the footprint and indicate DNA bending induced by protein binding.
Hydroxyl Radical Footprinting
Hydroxyl radical footprinting uses the highly reactive hydroxyl radical to cleave DNA backbone at every position with little sequence bias. The radical is generated by the Fenton reaction using iron-EDTA, ascorbate, and hydrogen peroxide. Because the radical is smaller than DNase I, hydroxyl radical footprinting provides higher resolution, detecting contacts on individual nucleotide faces. It is particularly useful for analyzing the periodicity of protein-DNA interactions along a DNA helix.
In Vivo Approaches
In vivo footprinting uses agents that modify DNA in living cells. Dimethyl sulfate methylates guanine residues that are not protected by bound proteins. After treatment, DNA is isolated, cleaved at methylated sites, and analyzed by ligation-mediated PCR. Comparison of in vivo and in vitro patterns reveals which binding sites are occupied in the cell. Chromatin accessibility assays such as DNase-seq and ATAC-seq provide genome-wide views of open chromatin but at lower resolution than footprinting.
Applications
EMSA is used for confirming predicted transcription factor binding sites, characterizing mutant binding sites, studying cooperative interactions, and monitoring binding activity during purification. Footprinting identifies the exact DNA sequence recognized by a protein and reveals conformational changes induced by binding.
Limitations
EMSA requires relatively stable complexes that survive electrophoresis. Rapidly dissociating complexes may not be detected. The gel matrix can stabilize weak interactions but also create artifacts from caging effects. Both techniques require purified DNA probes and are not suitable for high-throughput analysis. Quantitative EMSA requires careful control of probe specific activity and detection linearity.
