**4.3 Binding reaction**

The interaction between proteins and nucleic acid is sensitive to salt concentration and pH as it will influence the protein charge and conformation. However, the experimental conditions are very versatile in that different buffers can achieve good results. The most commonly used are Tris based buffers but other options include 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)-propanesulfonic acid (MOPS), and glycine or phosphate buffers. Naturally, it is advisable to provide an environment as close as possible to physiological conditions so the data obtained *in vitro* can be related to what happens *in vivo*.

Additives can be included in the binding reaction either if the interactions require the presence of co-factors or stabilizing agents, or as helpful components to minimize nonspecific binding. Glycerol or other small neutral solutes, for example sucrose, can be added to the binding mixture to stabilize labile proteins or enhance the stability of the interaction (Vossen et al., 1997). These solutes are used at final concentrations of 2M or less, as higher concentrations might interfere with the sample's viscosity and complicate handling. Other assays may require the presence of co-factors for a correct interaction such as the presence of cAMP for the *E. coli* CAP protein (Fried & Crothers, 1984) or ATP for human recombinase Rad51 (Chi et al., 2006). Non-ionic detergents are used to maximize protein solubility. In this case, the concentrations used depend on the detergent and system under study. Nuclease and phosphatase inhibitors can be useful as well as protease inhibitors, which as mentioned before, are particularly important when the protein sample comes from cell extracts. These inhibitors are commercially available and the concentration depends on the manufacturer's instructions. Some of the additives mentioned, particularly those involved in stabilizing the formed complexes can be included not only in the binding mixture but also in the gel buffers.

To minimize non-specific loss of protein the addition of a carrier protein (less than 0.1mg/mL) such as bovine serum albumin can be very helpful. The addition of unlabeled competing nucleic acids is suitable when there are secondary binding activities that mask the relevant one. Of course this only works if the protein interacts with the target nucleic acid with greater affinity then its competitor and the secondary binding does not discriminate between the sequences. Since the presence of a competing nucleic acid will always reduce the amount of specific binding, testing different competitors and concentrations is needed to optimize the assay. Another option to circumvent the problem of non-specific binding is the addition of salt at concentrations that will disrupt non-specific ionic bonds but leave the more specific interactions unimpaired.

#### **4.4 Non-denaturing gel electrophoresis**

After the binding reaction the free nucleic acid is separated from the formed complexes by non-denaturing gel electrophoresis. EMSA can be performed on polyacrylamide or agarose gels depending mainly on the size of the nucleic acid and desired resolution. The average pore size is estimated to be around 5 to 20nm in diameter for 10 and 4% acrylamide gels respectively (Lane et al., 1992). Typically the higher concentration gels are used for oligonucleotides and small RNAs and the lowest concentration for DNA fragments of around 100bp. A polyacrylamide gradient gel is sometimes preferred over linear gels as the gradient in pore size increases the range of molecular weight fractioned in a single run, which is particularly important when the complex has a much higher weight than the free nucleic acid (Walker, 1994). When complexes of different composition are formed, the gradient gels are also more likely to separate those with close molecular weight.

Agarose gels, on the other hand, have a pore size of around 70 to 700nm (Lane et al., 1992) in diameter and are therefore mostly used in assays with larger nucleic acid fragments or when large protein complexes are expected. Overall, polyacrylamide gels offer a better resolution for nucleic acid-protein complexes with a molecular weight of up to 500,000Da (Fried, 1989 as cited in Hellman & Fried, 2007).

Regarding the electrophoresis buffers, it should be taken into account the fact that the interaction between nucleic acids and proteins involves an ionic component. Therefore, the buffer's ionic strength and pH are important features that play a role in the complex stability. Although this is a very important factor there hasn't been, to our knowledge, any thorough study on the subject. The choice of electrophoresis buffers is varied and generally low ionic strength buffers are preferred and sometimes coincide with the buffer used in the binding reaction. Buffers with a medium salt concentration help stabilize the complexes, generate less heat during electrophoresis and also increase the speed of migration. High salt concentrations not only disrupt the complexes but also interfere with its movement into the gel matrix and lead to significant heating during the electrophoresis. Too low salt concentrations can also disrupt the stability of the preformed complexes as well as separate a double stranded DNA template (Kerr, 1995). The most common buffers are TBE (90mM Tris-Borate, 2mM EDTA, pH 8) and TAE (40mM Tris-Acetate, 1mM EDTA, pH 8). However, there are some complexes that cannot be detected with the classical buffers. For example the complexes formed between phage Mu repressor and its operators have an electrophoresis buffer-dependent stability and require Tris-glycine buffer at pH 9.4. (Alazard et al., 1992 as cited in Lane et al., 1992).

Particularly, in agarose gels it is important to monitor the temperature during electrophoresis to prevent the gel from heating up which could result in dissociation of the nucleic acid-protein complexes. Some cases may require that pre-cooling of the gel or even that the electrophoresis proceeds at lower than room temperatures, which can be achieved with special refrigeration devices.

#### **4.5 Detection**

214 Gel Electrophoresis – Advanced Techniques

On the other hand, the longer nucleic acid targets avoid these problems but will have more non-specific binding sites and the mobility shift is generally smaller requiring longer electrophoresis times as they run more slowly. A compromise needs to be reached

The interaction between proteins and nucleic acid is sensitive to salt concentration and pH as it will influence the protein charge and conformation. However, the experimental conditions are very versatile in that different buffers can achieve good results. The most commonly used are Tris based buffers but other options include 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)-propanesulfonic acid (MOPS), and glycine or phosphate buffers. Naturally, it is advisable to provide an environment as close as possible to physiological conditions so the data obtained *in vitro* can be related to

Additives can be included in the binding reaction either if the interactions require the presence of co-factors or stabilizing agents, or as helpful components to minimize nonspecific binding. Glycerol or other small neutral solutes, for example sucrose, can be added to the binding mixture to stabilize labile proteins or enhance the stability of the interaction (Vossen et al., 1997). These solutes are used at final concentrations of 2M or less, as higher concentrations might interfere with the sample's viscosity and complicate handling. Other assays may require the presence of co-factors for a correct interaction such as the presence of cAMP for the *E. coli* CAP protein (Fried & Crothers, 1984) or ATP for human recombinase Rad51 (Chi et al., 2006). Non-ionic detergents are used to maximize protein solubility. In this case, the concentrations used depend on the detergent and system under study. Nuclease and phosphatase inhibitors can be useful as well as protease inhibitors, which as mentioned before, are particularly important when the protein sample comes from cell extracts. These inhibitors are commercially available and the concentration depends on the manufacturer's instructions. Some of the additives mentioned, particularly those involved in stabilizing the formed complexes can be included not only in the binding

To minimize non-specific loss of protein the addition of a carrier protein (less than 0.1mg/mL) such as bovine serum albumin can be very helpful. The addition of unlabeled competing nucleic acids is suitable when there are secondary binding activities that mask the relevant one. Of course this only works if the protein interacts with the target nucleic acid with greater affinity then its competitor and the secondary binding does not discriminate between the sequences. Since the presence of a competing nucleic acid will always reduce the amount of specific binding, testing different competitors and concentrations is needed to optimize the assay. Another option to circumvent the problem of non-specific binding is the addition of salt at concentrations that will disrupt non-specific

After the binding reaction the free nucleic acid is separated from the formed complexes by non-denaturing gel electrophoresis. EMSA can be performed on polyacrylamide or agarose

ionic bonds but leave the more specific interactions unimpaired.

depending on what the EMSA study is trying to achieve.

**4.3 Binding reaction** 

what happens *in vivo*.

mixture but also in the gel buffers.

**4.4 Non-denaturing gel electrophoresis** 

The detection of an EMSA result will naturally depend on the labels used if any has been used. The results uncovered can involve the detection of the mobility shift between free nucleic acid and the complexed form or the detection of the mobility shift of free protein and the complexes.

Looking at the nucleic acid component without any label added the shift in mobility can be detected by staining with molecules that bind nucleic acids. Different products can be used

Electrophoretic Mobility Shift Assay: Analyzing Protein – Nucleic Acid Interactions 217

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

Fig. 3. Titration of a 500bp DNA fragment with the small delta antigen to estimate binding

Electrophoresis was in a 1.5% agarose gel in TAE buffer and the DNA was stained with

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

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.

one protein in the presence or absence of the other(s).

constant is between 1 and 1.5µM.

ethidium bromide.

**5.2 Cooperativity** 

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 immunohistochemistry or chemiluminescence approaches.

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 but the mobility shift can be visualized by autoradiography.
