**3. How complexes migrate in gels**

In this section, we will start with a simple account of the characteristics of the electrophoretic mobility of nucleic acids alone, and afterwards we will discuss how the formation of protein-nucleic acid complexes alters these characteristics.

Electrophoretic Mobility Shift Assay: Analyzing Protein – Nucleic Acid Interactions 209

of the small delta antigen (Fig.1. well 2) to a 400bp DNA fragment results in the formation of a complex with decreased gel mobility when compared with the unbound DNA (Fig.1. well 1). We can conclude that under our *in vitro* binding conditions, the small delta antigen

It is expected that when protein binds a nucleic acid fragment there will be a decrease in relative mobility and if the protein doesn't induce any appreciable bend on the nucleic acid then the conformational contribution to the decrease is small. Although an increase in the protein molecular weight results in reduction of gel migration it has been reported that the increase of the nucleic acid length can have the opposite effect. This was reported for the Lac repressor bound to DNA fragments of increasing sizes, which resulted in an increase of relative mobility (Fried, 1989). This observation indicates that the ratio of protein and nucleic acid weights is more important in the migration than the absolute weight of the complex. Another interesting study reports that the binding of protein to a nucleic acid can accelerate mobility. This was observed for relatively large linear DNA binding to a protein from the hyperthermophilic *Methanothermus fervidus* that was shown to induce nucleic acid condensation (Sandman et al., 1990). In this case the conformational change of the DNA is a stronger factor than the weight increase, causing acceleration rather than a decrease in

Overall, the conformational features that influence gel migration of protein-nucleic acid complexes are not thoroughly studied and questions are only raised when exceptions emerge such as the ones mentioned above. Nowadays, the EMSA method is almost exclusively used to analyze the interaction between proteins and nucleic acids and to a lesser extent its conformations that can influence gel migration. When exceptions arise and the retardation pattern is not exactly as predicted, it can still point out clearly whether the molecules are interacting or not. In the end, the exact location of the resulting gel bands

External factors can also influence the separation of the bound or unbound nucleic acid such as the nature of the gel matrix and temperature during electrophoresis. Generally, the best resolution is obtained with the smallest pore diameter that allows the migration of unbound nucleic acid. However, if large complexes are expected there should be a compromise in pore size so that they can enter the gel matrix. As will be discussed below, polyacrylamide gels offer the best conditions for small complexes and nucleic acid fragments. On the other

The detection of a protein-nucleic acid complex within a gel depends critically on the resolution obtained between unbound nucleic acid and the formed complexes as well as its stability within the gel matrix. In most cases, the gel matrix is expected to stabilize the preformed complex as it impedes the diffusion of dissociating components maintaining the concentration of protein and nucleic acid (and complex) at levels as high or higher than those achieved in the equilibrium binding reaction. This of course is compromised if for instance the salt concentration in the binding reaction differs largely from that in the electrophoresis/gel buffer, resulting in an adjustment in salt concentration that could disrupt the complexes formed. As the gel retardation method is an *in vitro* assay, when extrapolating to the *in vivo* conditions one must be careful as the former may provide

favorable binding conditions that are not achieved at physiological concentrations.

cannot be predicted but the answer is usually unambiguous.

hand, agarose gels are more suitable for larger aggregates.

interacts with the given 400bp DNA fragment causing a clear mobility shift.

relative mobility.

In a non-denaturing agarose or polyacrylamide gel and conventional buffer conditions the nucleic acids, being negatively charged, will migrate towards the anode when electric current is applied. The gel will then act as a sieve selectively impeding the migration in proportion to the nucleic acid molecular weight, which is generally proportional to its charge. Therefore, and as the weight is approximately related to chain length, the length of nucleic acid is estimated by its migration. There is though another property that affects gel migration that is the topology of the nucleic acid (conformation, circularity) making the molecules seem longer or shorter than they really are. Secondary and tertiary structures can be removed using denaturing agents (for example, formaldehyde, formamide and urea) allowing for the electrophoretic mobility to become a simple function of molecular weight. Obviously, this denaturing step cannot be applied in a gel retardation assay as it would impede the interaction between the protein and nucleic acid.

Fig. 1. Example of an electrophoretic mobility shift assay. An unlabeled DNA of 400 base pairs (bp) was incubated in a phosphate buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.5mM KH2PO4, pH 7.4) in the absence (1) or presence (2) of 2µM of small delta antigen. The samples were loaded onto a 1.5% agarose gel and after electrophoresis in TAE buffer (40mM Tris acetate, 1mM EDTA) the DNA was stained with ethidium bromide. (M) represents the molecular weight marker (GeneRuler DNA Ladder mix, Fermentas).

When a protein is added to the mix and interacts with the nucleic acid forming complexes it results in a change in gel migration relative to that of the free nucleic acid. This shift is mainly due to an obvious increase in the molecular weight, the adjustment of charge and eventual changes in the nucleic acid conformation. In figure 1 we give an example of an EMSA study where the small delta antigen was added to DNA. It is clear that the addition

In a non-denaturing agarose or polyacrylamide gel and conventional buffer conditions the nucleic acids, being negatively charged, will migrate towards the anode when electric current is applied. The gel will then act as a sieve selectively impeding the migration in proportion to the nucleic acid molecular weight, which is generally proportional to its charge. Therefore, and as the weight is approximately related to chain length, the length of nucleic acid is estimated by its migration. There is though another property that affects gel migration that is the topology of the nucleic acid (conformation, circularity) making the molecules seem longer or shorter than they really are. Secondary and tertiary structures can be removed using denaturing agents (for example, formaldehyde, formamide and urea) allowing for the electrophoretic mobility to become a simple function of molecular weight. Obviously, this denaturing step cannot be applied in a gel retardation assay as it would

Fig. 1. Example of an electrophoretic mobility shift assay. An unlabeled DNA of 400 base pairs (bp) was incubated in a phosphate buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.5mM KH2PO4, pH 7.4) in the absence (1) or presence (2) of 2µM of small delta antigen. The samples were loaded onto a 1.5% agarose gel and after electrophoresis in TAE buffer (40mM Tris acetate, 1mM EDTA) the DNA was stained with ethidium bromide. (M) represents the molecular weight marker (GeneRuler DNA Ladder mix,

When a protein is added to the mix and interacts with the nucleic acid forming complexes it results in a change in gel migration relative to that of the free nucleic acid. This shift is mainly due to an obvious increase in the molecular weight, the adjustment of charge and eventual changes in the nucleic acid conformation. In figure 1 we give an example of an EMSA study where the small delta antigen was added to DNA. It is clear that the addition

impede the interaction between the protein and nucleic acid.

Fermentas).

of the small delta antigen (Fig.1. well 2) to a 400bp DNA fragment results in the formation of a complex with decreased gel mobility when compared with the unbound DNA (Fig.1. well 1). We can conclude that under our *in vitro* binding conditions, the small delta antigen interacts with the given 400bp DNA fragment causing a clear mobility shift.

It is expected that when protein binds a nucleic acid fragment there will be a decrease in relative mobility and if the protein doesn't induce any appreciable bend on the nucleic acid then the conformational contribution to the decrease is small. Although an increase in the protein molecular weight results in reduction of gel migration it has been reported that the increase of the nucleic acid length can have the opposite effect. This was reported for the Lac repressor bound to DNA fragments of increasing sizes, which resulted in an increase of relative mobility (Fried, 1989). This observation indicates that the ratio of protein and nucleic acid weights is more important in the migration than the absolute weight of the complex. Another interesting study reports that the binding of protein to a nucleic acid can accelerate mobility. This was observed for relatively large linear DNA binding to a protein from the hyperthermophilic *Methanothermus fervidus* that was shown to induce nucleic acid condensation (Sandman et al., 1990). In this case the conformational change of the DNA is a stronger factor than the weight increase, causing acceleration rather than a decrease in relative mobility.

Overall, the conformational features that influence gel migration of protein-nucleic acid complexes are not thoroughly studied and questions are only raised when exceptions emerge such as the ones mentioned above. Nowadays, the EMSA method is almost exclusively used to analyze the interaction between proteins and nucleic acids and to a lesser extent its conformations that can influence gel migration. When exceptions arise and the retardation pattern is not exactly as predicted, it can still point out clearly whether the molecules are interacting or not. In the end, the exact location of the resulting gel bands cannot be predicted but the answer is usually unambiguous.

External factors can also influence the separation of the bound or unbound nucleic acid such as the nature of the gel matrix and temperature during electrophoresis. Generally, the best resolution is obtained with the smallest pore diameter that allows the migration of unbound nucleic acid. However, if large complexes are expected there should be a compromise in pore size so that they can enter the gel matrix. As will be discussed below, polyacrylamide gels offer the best conditions for small complexes and nucleic acid fragments. On the other hand, agarose gels are more suitable for larger aggregates.

The detection of a protein-nucleic acid complex within a gel depends critically on the resolution obtained between unbound nucleic acid and the formed complexes as well as its stability within the gel matrix. In most cases, the gel matrix is expected to stabilize the preformed complex as it impedes the diffusion of dissociating components maintaining the concentration of protein and nucleic acid (and complex) at levels as high or higher than those achieved in the equilibrium binding reaction. This of course is compromised if for instance the salt concentration in the binding reaction differs largely from that in the electrophoresis/gel buffer, resulting in an adjustment in salt concentration that could disrupt the complexes formed. As the gel retardation method is an *in vitro* assay, when extrapolating to the *in vivo* conditions one must be careful as the former may provide favorable binding conditions that are not achieved at physiological concentrations.

Electrophoretic Mobility Shift Assay: Analyzing Protein – Nucleic Acid Interactions 211

on the nature of the protein, it is advisable to isolate nuclear and cytoplasmic proteins from crude extracts improving the results. Particularly, if the binding protein is thought to be nuclear and in low abundance, the isolation of nuclear extracts will prevent the dilution that would occur if whole cell extracts were used, which could render the concentration too low

Cell extracts are easy and relatively fast to obtain and the methods are commonly derived from the protocol described by Dignam and collaborators almost three decades ago (Dignam et al., 1983). This method isolates both nuclear and/or cytoplasmic proteins suitable for later analysis using EMSA. One disadvantage in preparing cell extracts is its crudeness; they generally degrade faster than purer preparations due to the presence of cellular proteases. To limit protein degradation or alteration the protocol should be performed on ice or at 4ºC and protease inhibitors should be added. A control test can easily be performed to assess the viability of the extract by using ubiquitous DNA probes (Kerr, 1995). If these fail than the cell extract might be "dead". Despite its disadvantages cell extracts are needed when the interest lies in identifying new nucleic acid-binding proteins or when a complex of different proteins is needed to interact with the target nucleic acid as sometimes one recombinant protein cannot bind by itself. Tissue samples can also be a source of protein sample for these assays. The same care should be taken as in whole cell

If the nucleic acid-binding protein is known then recombinant proteins can be expressed and purified. Recombinant or heterologous proteins are commonly expressed in bacteria or an eukaryotic cell line of interest. Fusion proteins of the target are generally constructed with a tag to facilitate purification. Common tags, such as glutathione-S-transferase (GST), tandem affinity purification tag (TAP tag), maltose binding protein (MBP) or 6xHistidine, are cloned in frame with the protein. Sometimes it is possible to include a protease cleavage site between the protein of interest and the tag so the latter can be easily removed after purification. Even though a tag can be very helpful, it should be taken into account that it can alter the recombinant protein conformation and even disrupt its binding ability. On the other hand they can be helpful in stabilizing the protein terminus they are close to. A careful study is needed when choosing the tag and usually small peptides are preferred to

There are several systems available for the production of heterologous proteins of which bacterial extracts of *Escherichia coli* are one of the most widely used. This Gram-negative bacterium remains an attractive host due to its ability to grow rapidly and with high density using inexpensive substrates. Its genetics has been well characterized for quite some time and there is a wide range of cloning vectors as well as mutant host strains that make it such a versatile system. Typically, the heterologous complementary DNA is cloned into a compatible plasmid which is then transfected into the bacteria to achieve a high gene dosage. This doesn't necessarily guarantee the accumulation of high levels of a full-length active form of the recombinant protein but other efforts can be made to improve that. To achieve high-level production in *E. coli* strong promoters should be used such as the bacteriophage T7 late promoter, and usually the T7 polymerase is also present under IPTG (isopropyl-β-D-1-thiogalactopyranoside)-induction. In the past years several strains have been engineered to improve the recombinant protein yields through efforts to increase mRNA stability as well as improve transcription

for the protein to be even detected.

extracts to minimize the activity of proteases.

minimize its impact on the recombinant protein of interest.
