**5. EMSA applications**

The gel retardation assay has been used under different conditions in order to achieve specific results. The method is useful in studying not only the interaction between proteins and nucleic acids but also in assessing nucleic acid conformational characteristics. It can be used to characterize bends in the DNA double helix with polyacrylamide gels and comparative measurements (for an example Crothers & Drak, 1992) or to detect complexes formed with super coiled DNA being sometimes designated as topoisomer gel retardation (for examples see Palecek, 1997; Nordheim & Meese**,** 1988). In this section we mention how a gel retardation assay can help characterize protein-nucleic acid interactions.

#### **5.1 Binding constants**

Although EMSA is most commonly used as a qualitative assay it can, under certain conditions, provide quantitative data for relatively stable complexes. One of its earliest applications was in the measurement of kinetic and thermodynamic parameters. The association rates are determined by mixing the complex components at known concentrations and loading them in a running gel at precise intervals (for an example Spinner et al., 2002). For dissociation rates, a time course experiment is done by addition of competing nucleic acid to the preformed complexes (Fried & Crothers, 1981). The binding constant can be determined by the amount of complex formed as a function of protein concentration at equilibrium or as a ratio of the association and dissociation constants (for an example Demarse et al., 2009). An alternative method to measure kinetic and thermodynamic constants is the nitrocellulose filter binding assay that will be mentioned below.

As an example we show in figure 3 the titration of a DNA with the small delta protein to assess binding constants. The binding reaction was done by incubating the samples in a phosphate buffer during the same period of time (10 minutes) and then loading them onto an agarose gel for electrophoresis. It is clear that when the protein is present at only 0.25µM it does not interfere with the DNA mobility (Fig.3. well 2) as the band covered the same distance as the first sample, in which the protein was not present (Fig.3. well 1). But when 1.5µM of the small delta antigen are present in the binding reaction there is almost no free DNA present and the majority of the molecules are bound in a complex (Fig3. well 5). In the intermediate concentrations it can be clearly observed the decreasing presence of free DNA and increasing DNA-protein complexes as the protein concentration raises. We can consider that the dissociation constant can be estimated by quantifying the disappearance of the free DNA band (Demarse et al., 2009). From figure 3 we can say that the apparent dissociation constant is between 1 and 1.5µM.

Fig. 3. Titration of a 500bp DNA fragment with the small delta antigen to estimate binding constants. An unlabeled 500bp DNA complementary to part of the HDV RNA was incubated, in a phosphate buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.5mM KH2PO4, pH 7.4), with increasing concentrations of small delta antigen of 0; 0.25; 0.5; 1; and 1.5µM and samples were loaded onto wells 1, 2, 3,4 and 5, respectively. Electrophoresis was in a 1.5% agarose gel in TAE buffer and the DNA was stained with ethidium bromide.

#### **5.2 Cooperativity**

216 Gel Electrophoresis – Advanced Techniques

ranging from the classic but hazardous ethidium bromide to other chromophores or fluorophores such as RedSafe DNA Stain (ChemBio) or SYBR® Safe DNA gel stain (Invitrogen). When the nucleic acid has been previously labeled the detection methods depend on the nature of the label. A 32P radioisotope is one of the easiest and most sensitive methods to detect nucleic acids but it's a hazardous material to work with. Other very common labels are biotin, digoxigenin or fluorophores. These labels are innocuous but usually give less sensitive results and the detection procedure can involve extra steps such as transfer to a membrane and incubation with primary and secondary antibodies as well as intermediate washing steps. The results in these cases can be observed by

The detection of protein mobility shift involves less direct methods, meaning, extra steps such as a denaturing step and electrotransfer onto a membrane, may be necessary as they are usually immunodetected. If the protein of interest is known, and a specific antibody is available, it can be used in detection. If not, a method such as the one discussed above, proposed by Adachi and colleagues that involves labeling the thiol group of cysteins and using an antibody against the label. Stepwise, the easier way to detect protein in an EMSA is by labeling it with radioisotope, a method designated by reverse EMSA that will be discussed ahead. This procedure has the disadvantage of working with radioactive material

The gel retardation assay has been used under different conditions in order to achieve specific results. The method is useful in studying not only the interaction between proteins and nucleic acids but also in assessing nucleic acid conformational characteristics. It can be used to characterize bends in the DNA double helix with polyacrylamide gels and comparative measurements (for an example Crothers & Drak, 1992) or to detect complexes formed with super coiled DNA being sometimes designated as topoisomer gel retardation (for examples see Palecek, 1997; Nordheim & Meese**,** 1988). In this section we mention how

Although EMSA is most commonly used as a qualitative assay it can, under certain conditions, provide quantitative data for relatively stable complexes. One of its earliest applications was in the measurement of kinetic and thermodynamic parameters. The association rates are determined by mixing the complex components at known concentrations and loading them in a running gel at precise intervals (for an example Spinner et al., 2002). For dissociation rates, a time course experiment is done by addition of competing nucleic acid to the preformed complexes (Fried & Crothers, 1981). The binding constant can be determined by the amount of complex formed as a function of protein concentration at equilibrium or as a ratio of the association and dissociation constants (for an example Demarse et al., 2009). An alternative method to measure kinetic and thermodynamic constants is the nitrocellulose filter binding

As an example we show in figure 3 the titration of a DNA with the small delta protein to assess binding constants. The binding reaction was done by incubating the samples in a

a gel retardation assay can help characterize protein-nucleic acid interactions.

immunohistochemistry or chemiluminescence approaches.

but the mobility shift can be visualized by autoradiography.

**5. EMSA applications** 

**5.1 Binding constants** 

assay that will be mentioned below.

Proteins can bind nucleic acids in a cooperative manner, that is, the complexes formed involve the binding of more than one protein to a specific nucleic acid segment. These multiprotein complexes may be a consequence of direct protein-protein interaction needed for nucleic acid binding, or a protein-induced deformation of the nucleic acid is a prerequisite to facilitate the binding of a second protein, or it may result from the bringing together of molecules bound at distinct sites in the nucleic acid sequence. The cooperativity can be inferred in a gel retardation assay from the underrepresentation of intermediate complexes between the unbound and saturated states. Multiprotein complexes can be comprised of a single protein species forming a homomultimer or of different proteins. The latter can be easily characterized by EMSA by the stability of the complexes formed with one protein in the presence or absence of the other(s).

Electrophoretic Mobility Shift Assay: Analyzing Protein – Nucleic Acid Interactions 219

The multiplexed EMSA was developed in 2008 by Smith and Humphries to characterize nuclear protein and DNA interactions, namely with transcription factors. In this method the nuclear extract is incubated with a pool of unlabeled DNA consensus competitors prior to adding the labeled DNA probe. An initial EMSA run will determine which cocktail competes with the probe binding to nuclear proteins which will then run individually in another EMSA to determine the precise competitor (Smith & Humphries, 2008). It is a competition-based method to identify uncertain DNA binding proteins requiring only a

The two-dimensional EMSA is a process that combines EMSA with proteomic or sequencing techniques to identify the proteins or the nucleic acid sequences that are present in the formed complexes. Two slightly different protocols have been developed to identify the

An initial approach was proposed by Woo and colleagues as they tried to identify and characterize transcription factors (Woo et al., 2002). A crude nuclear extract is partially purified by gel filtration and the resulting fractions are then bound to the nucleic acid probe and analyzed by EMSA. Meanwhile, in parallel, the pI and molecular weight of the putative interacting protein(s) is estimated as the fractions are analyzed by isoelectric focusing or SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) in order to characterize possible candidates. Next, spots with the predetermined pI and molecular weight of the candidates are excised from a two-dimensional array of nuclear proteins and the proteins are eluted, renatured and tested for their binding ability through EMSA and the spots are afterwards analyzed by mass spectrometry for protein identification. This method is limited to proteins that can re-form into functional nucleic acid-binding conformations after the denaturing SDS-PAGE step, although EMSA can still show results even if renaturation efficiency is low. Because the final EMSA step that confirms the binding is performed with protein eluted from single spots it is only possible to identify proteins that interact with the nucleic acids as monomers or homomultimers. Proteins that only interact when complexed with other

A similar 2D-EMSA technique has since then been developed that incorporates EMSA into a two-dimensional proteomics approach by replacing the isoelectric focusing with EMSA as the first dimension of the 2D method (Stead et al., 2006). The protein sample, in the presence or absence of the nucleic acid, is separated by native PAGE as in a conventional EMSA. The protein bands from both conditions are then separated in a second dimension by denaturing SDS-PAGE. The proteins showing the nucleic acid dependent shift in mobility can be extracted from the gel for mass spectrometry identification. This approach does not require any previous knowledge of the chemical or physical properties of the binding protein and does not require protein renaturation after gel excision. It is also not limited to identify proteins that bind by themselves or as homomultimers and allows the characterization of

These 2D approaches were developed by the two groups to study transcription factors, therefore, double stranded DNA is used as a nucleic acid probe but they can also adapted to

interacting proteins and another method aims at the target nucleic acid sequence.

**6.3 Multiplexed competitor EMSA (MC-EMSA)** 

**6.4 Two-dimensional EMSA (2D-EMSA)** 

prior knowledge of transcription factor consensus sequences.

proteins will give a negative result on the validation EMSA.

complexes composed of different proteins.
