Beginner’s guide to investigating protein: DNA interactions using electrophoretic mobility shift assays (EMSAs)

In 1962, Zuckerandl and Pauling stated that life is a relationship between molecules and not a property of any molecule, so is therefore disease. This profound statement succinctly describes the biochemistry of life. At the core of these interactions lies the interaction between DNA and proteins in a crowded cellular environment.

Using biochemistry to understand protein–DNA interactions has been a long-pursued scientific endeavor. This article focuses on using simple biochemical techniques that can give us deep insight into how well proteins can bind and interact with DNA.

One of the most common ways to visualize protein–DNA binding is using the simplicity of DNA migrations. Nucleotides form phosphodiester bonds between themselves to form DNA. These phosphodiester bonds result in the presence of a negatively charged phosphate group. The presence of these phosphate groups and a free negative charge makes DNA negatively charged in nature. In a gel–matrix environment powered by electricity, DNA will migrate towards a positively charged electrode. This characteristic of DNA is exploited in understanding its ability to bind protein. Electrophoresis of DNA is performed in two states: native and denaturing. In the denaturing state, the components are denatured using heat or chemicals and passed through a matrix that also denatures them. This essentially eliminates shape effects of these biomolecules and allows migration based on size. However, this will not account for interactions or secondary/tertiary structures that arise in proteins. In the native conditions, the components are investigated as such, allowing investigation of complexes. DNA alone migrates faster than DNA that is bound by a protein. Thus, the migration allows scientists to determine interactions between these core biomolecules (Figure 1). This article describes the methods used to perform an electrophoretic mobility shift assay (EMSA), a robust and sensitive method to determine protein–DNA binding (Figure 1).

• DNA (double-stranded or single-stranded depending on the experiment and the characteristic of the protein) – DNA is typically labeled in order to visualize it separately from the background. This can either be a fluorescent label or a radioactive isotope, typically ³²P. Labeled DNA allows usage in limited quantities, which makes the calculations of binding affinities more accurate. As an alternative, the gel can be post-stained with a nucleic acid dye such as SYBR GOLD or SYBR SAFE. Unlabeled DNA, however, must meet the detection limit of the dyes used for staining. The disadvantage with post staining is that it can be noisy especially when studying ribonucleic protein interactions with DNA such as that for CRISPR proteins. This is because RNA also gets stained by these nucleic acid dyes and can be counterintuitive, but this can be overcome by running controls with the individual components for comparison. A wide range of sizes can be used in an EMSA, as well as linear and circular DNA.

• Protein – Fully or partially pure protein can be used. A protein titration is recommended with limiting DNA concentrations in order to generate a binding curve. Based on the curve, the exact affinity of interaction and kinetic parameters can be calculated.

• Acrylamide – Bis-acrylamide is used to make native gels without denaturing agents (ratio depends on protein size, typically 29:1 is used for polymerization and the size will vary depending on the requirements of the experiment and the apparatus). The gel percentage is also decided depending on the size of DNA that needs to be resolved.

• Buffer – A good binding buffer is necessary to allow interaction between protein and DNA. This typically contains a buffer component such as TRIS or HEPES, along with a monovalent salt. Some assays also require multivalent ions for protein function. The conductivity of the sample and the buffer needs to be matched for a successful electrophoresis run.

  • Polyethylene glycol can be added to the buffer to improve entry into the gel. This does not affect the resolution of electrophoresis.

• Gel plates and apparatus – to pour gels and run them. Precast gels obtained from a manufacturer can also be used. Gel percentage needs to be determined based on the size of the products being resolved on the gel.

The conditions described in the protocol generally work for most proteins but if binding is not detected, steps can be taken to troubleshoot and optimize the protocol. Typically, the concentrations of the protein and nucleic acids, degradation of the components or inefficient labeling techniques can cause the absence of bands. Uneven gel polymerization, overheating and high sample conductivity can cause smeared bands. Aggregate proteins and a high protein:DNA ratio can cause the complex to be stuck in wells. Proteins have unique features that facilitate diverse migration patterns in gels. Keeping this in mind, there are a few other components that can be optimized to achieve the best results with EMSA. Here are a few options to consider:

1. Equilibration buffer – It is important to use a buffer with the correct pH. This will depend on the isoelectric point of the protein (pH at which the protein carries no net electrical charge) and will determine how well the protein complex is able to migrate within the gel matrix.

2. Temperature at which protein and DNA are equilibrated – Temperature affects the binding affinity of ligands to substrates. It is important to try various temperatures to ensure that the best temperature is chosen for pursuing further binding studies.

3. Stoichiometry of protein:DNA in the equilibration reactions (concentration of protein and DNA) – Technically, for a good binding experiment, the concentration of the protein should not affect the KD value. In terms of protein–DNA binding, KD is the concentration at which half the DNA is bound to the protein. Lower the KD, higher the affinity of binding. However, it is also important to use a limiting concentration of the substrate with respect to the protein to ensure that the binding sites in the protein are not saturated.

4. Acrylamide:bis-acrylamide (ratio depends on protein size, typically 29:1 is used) percentage for better resolution/separation. A gradient gel can also be used. The percentage of the gel varies between 4% and 20% and as the percentage increases, the resolution and separation between bands increase. The gel percentage also determines how well the complexes migrate from the wells into the gel.

Native gels are prepared without denaturing reagents. Gels are generally made using acrylamide:bis-acrylamide. Meanwhile, set up the reaction along with controls including DNA, protein, and a ladder to measure sizes after electrophoresis. The temperature at which the binding reaction occurs is usually important and needs to be optimized; most reactions are performed at 4°C, room temperature, or 37°C. A titration of the protein is usually performed. You need to ensure that the volume remains constant in all reactions, while altering the concentration of the protein to set up a titration. Equilibration time depends on the components and are dependent on temperature, pressure, and salt concentrations. Usually, most reactions are performed for 30 minutes or 60 minutes and then optimized based on the preliminary results. It is recommended to do the experiment for longer incubation times to ensure that the reaction reaches equilibrium. An accurate binding experiment would determine KD values that are not dependent on DNA concentrations and includes controls that can reduce artifacts. The gel is usually run in colder conditions to prevent it from warming up and denaturing the components whilst electrophoresing. The voltage can be reduced if the gel becomes warm. After the run, the gel can be visualized using appropriate equipment. For instance, Typhoon gel scanner can be used to visualize fluorescently labeled DNA and radioactively labeled DNA.

EMSA is a robust and versatile technique that can be applied in various fields of biological research. It can be used to qualitatively study protein:DNA interactions and determine binding affinity. Some of the applications are as follows:

1. EMSA can be used to identify protein:DNA interactions. This is useful to understand how some proteins interact with DNA and how these interactions can affect protein function. This is especially useful in the case of DNA-binding proteins.

2. EMSA is also used to map DNA-binding sites of a protein on a piece of DNA. Mutating sites on a DNA fragment can also shine light on the sequence specificity of the protein. Competition reactions can be set to determine protein specificity and its affinity for non-specific DNA.

3. The exact KD values can be determined and used to compute kinetic parameters.

4. Small molecules and drugs that affect protein:DNA interaction can be investigated through EMSA.

5. Post-translational modifications on protein:DNA interactions can be studied by using different protein variants.

1. Positive control: consists of a sample that will definitively give results in a binding experiment. This usually includes the same DNA probe with a protein that is known to bind it.

2. Negative control: consists of a sample that contains a protein that will definitively not bind to the DNA probe of interest. A DNA-only sample is also included to give a sense of how the free DNA will migrate in the absence of a protein.

3. Competitor control: consists of a DNA probe that is non-labeled and contains the same binding site as the labeled probe, added to a reaction containing the protein and the labeled probe. This will determine how specifically the protein binds to the probe of interest.

4. Antibody control: an antibody control is sometimes used, if the protein being investigated has antibodies available. This ensures that the protein:DNA complex being observed indeed contains the correct protein.

EMSA is a reliable and easy way to qualitatively check for protein–DNA binding. It uses reagents that are commonly present in biochemistry labs and provides a lot of information about the intricacies of binding events. While EMSA can be tweaked to suit the needs of the experiment, it has some limitations because of it is low-throughput readout. There are other methods that can be used as alternatives for EMSA that are listed below.

• Chromatin immunoprecipitation: Used to determine the association of specific proteins with specific genomic regions.

• Fluorescence polarization: useful for measuring binding affinities in a high-throughput manner. It also has high resolution since a low concentration of the DNA can be used to measure KD.

• DNA footprinting: Can be used to identify protein binding sites and identification of nucleic acids at or near these sites. Assay is performed under equilibrium conditions for the protein of interest unlike EMSA.

• Förster resonance energy transfer: Can offer single-cell resolution and elucidate conformational interactions between protein and DNA.

• Hellman, L.M. and Fried, M.G. (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat. Protoc. 2, 1849–1861. DOI: 10.1038/nprot.2007.249

• (2005) Electrophoretic mobility shift assays. Nat. Methods2, 557–558. DOI: 10.1038/nmeth0705-557

• Zuckerkandl, E. and Pauling, L. (1965) Molecules as documents of evolutionary history, J. Theor. Biol. 8, 357–366. DOI: 10.1016/0022-5193(65)90083-4

• Gel Shift Assays–EMSA. Protein Biology Resource Library. Available at: 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 [Accessed 5 September 2024]

Shravanti Suresh is a scientist at Mammoth Biosciences who works on characterizing CRISPR proteins and their interactions with nucleic acids. She completed her PhD in Biochemistry, investigating the mechanisms of CRISPR-Cas immune systems in bacteria and how they defend themselves against phages. Email: [email protected].