Electrophoresis through polyacrylamide gels (PAGE) is one of the most commonly used techniques for characterizing proteins and nucleic acids. It is especially useful to monitor changes in their conformations because the size and shape of a molecules are two of the main determinants of electrophoretic mobility through sieving gels (the other is the net charge). Electrophoresis, however, is primarily a comparative technique because little information is obtained solely from the mobility of a macromolecule under any single set of conditions. It is most informative when a change in mobility is observed. For example, unfolding of a protein or nucleic acid is apparent by a dramatic decrease in electrophoretic mobility. Phenomena whose conformation is induced to change under the influence of some agent, such as a denaturant, are readily studied by electrophoresis in transverse gradient gels. In such gels, the perturbing agent is included in the gel in a continuous gradient, perpendicular to the direction of electrophoretic mobility. In a sample applied uniformly across the top of such a gel, each molecule migrates under constant conditions but different from those adjacent to it on the gel. The population of molecules migrates under continuously varying conditions. Changes in the macromolecular structure at any point within the gradient are apparent from the change in mobility and give a characteristic shape to the electrophoretic band after electrophoresis. Such changes are most apparent if they occur abruptly, such as a cooperative change from form A to form B over a small part of the transverse gradient, when form A predominates at one extreme of the gradient and form B at the other extreme.
TGGE has the many advantages of electrophoretic techniques. The method is simple, rapid, and requires no expensive equipment, only small quantities of sample are required, and the sample need not necessarily be homogeneous. Although only semiquantitative, TGGE has the advantage that close comparisons can be made of different macromolecules by running them on the same gel. Even small differences in their electrophoretic mobilities are detected.
When a macromolecule interconverts between two forms, say AoB, as a result of the transverse gradient, a major question is whether one or two electrophoretic bands are observed at positions where the two species coexist. Numerical simulations indicate that this depends almost exclusively on the rate at which the two states are interconverted, relative to the duration of the electrophoretic separation (Fig. 1). If their interconversion is very slow, individual bands corresponding to both A and B are present to the extent that they were both present in the original sample. At the other extreme, with very rapid rates of interconversion and a half-time less than about 0.1 the duration of the electrophoretic separation, a single band will be present that has an intermediate electrophoretic mobility at the weighted average of the two. In this case, a change from one state to another at some point in a transverse gradient gel is apparent after the electrophoresis as a sigmoidal change in the mobility of a single, continuous band across the gradient. Then the final pattern describes the equilibrium between the two forms as a function of the variable in the gradient. With intermediate rates of interconversion, the electrophoretic patterns are smeared between the two positions of the two states A and B as various molecules convert to the other state at various times during the separation.
Figure 1. Simulated TGGE patterns expected for a macromolecule undergoing a two-state transition between forms A and B at four different rates and starting with the sample initially as either all A (left) or all B (right.) The transverse gradient was imagined to favor the A state on the left and the B state on the right, and the apparent equilibrium constant [A]/[B] varies logarithmically across the gradient from 102 to 10-2. It was assumed that both the forward and reverse rate constants vary inversely across the gradient by a factor of 10 2 The rate of the interconversion is at a minimum at the middle of the transverse gradient, and the half-time is tx / 2. The rate there is expressed on the left as the ratio of t x / 2 to the duration of the electrophoretic separation ( telect). Electrophoretic migration is from top to bottom, and state A has greater mobility. The distribution of the macromolecule in the final gel is given by the contours. With the most rapid interconversion (top), the final pattern is a single, continuous band that describes the equilibrium between A and B across the gradient (dashed sigmoidal curve). The patterns are the same, irrespective of whether form A or B is applied to the gel. With a slower transition, where the interconversion takes place with a half-time comparable to the duration of the electrophoretic separation, the continuous band is replaced by a smear because the initial molecules convert to the other form only once and at various times during the electrophoresis. The form applied to the gel persists increasingly with a slower transition. In the limit of a very slow transition, only the form applied to the gel would be present after the electrophoretic separation.
![Simulated TGGE patterns expected for a macromolecule undergoing a two-state transition between forms A and B at four different rates and starting with the sample initially as either all A (left) or all B (right.) The transverse gradient was imagined to favor the A state on the left and the B state on the right, and the apparent equilibrium constant [A]/[B] varies logarithmically across the gradient from 102 to 10-2. It was assumed that both the forward and reverse rate constants vary inversely across the gradient by a factor of 10 2 The rate of the interconversion is at a minimum at the middle of the transverse gradient, and the half-time is tx / 2. The rate there is expressed on the left as the ratio of t x / 2 to the duration of the electrophoretic separation ( telect). Electrophoretic migration is from top to bottom, and state A has greater mobility. The distribution of the macromolecule in the final gel is given by the contours. With the most rapid interconversion (top), the final pattern is a single, continuous band that describes the equilibrium between A and B across the gradient (dashed sigmoidal curve). The patterns are the same, irrespective of whether form A or B is applied to the gel. With a slower transition, where the interconversion takes place with a half-time comparable to the duration of the electrophoretic separation, the continuous band is replaced by a smear because the initial molecules convert to the other form only once and at various times during the electrophoresis. The form applied to the gel persists increasingly with a slower transition. In the limit of a very slow transition, only the form applied to the gel would be present after the electrophoretic separation.](http://what-when-how.com/wp-content/uploads/2011/05/tmp2414_thumb1.jpg "Simulated TGGE patterns expected for a macromolecule undergoing a two-state transition between forms A and B at four different rates and starting with the sample initially as either all A (left) or all B (right.) The transverse gradient was imagined to favor the A state on the left and the B state on the right, and the apparent equilibrium constant [A]/[B] varies logarithmically across the gradient from 102 to 10-2. It was assumed that both the forward and reverse rate constants vary inversely across the gradient by a factor of 10 2 The rate of the interconversion is at a minimum at the middle of the transverse gradient, and the half-time is tx / 2. The rate there is expressed on the left as the ratio of t x / 2 to the duration of the electrophoretic separation ( telect). Electrophoretic migration is from top to bottom, and state A has greater mobility. The distribution of the macromolecule in the final gel is given by the contours. With the most rapid interconversion (top), the final pattern is a single, continuous band that describes the equilibrium between A and B across the gradient (dashed sigmoidal curve). The patterns are the same, irrespective of whether form A or B is applied to the gel. With a slower transition, where the interconversion takes place with a half-time comparable to the duration of the electrophoretic separation, the continuous band is replaced by a smear because the initial molecules convert to the other form only once and at various times during the electrophoresis. The form applied to the gel persists increasingly with a slower transition. In the limit of a very slow transition, only the form applied to the gel would be present after the electrophoretic separation.")
If the starting macromolecule is homogeneous and all in one state, only one band should be apparent finally at each point in the gradient. Its mobility should be either that of the original state, if its interconversion is slow, or, if the interconversion is fast, of the final equilibrium mixture. The rates of such transitions can also be investigated more directly by comparing patterns obtained starting with the sample in either of the two states. Identical patterns are observed with rapid interconversions, but the original form persists if the interconversion is slow. If more than two states are possible, the resulting patterns may be more complex, but the same rules should apply to the interconversion of each pair of states.
The transverse gradient can be of any parameter that does not unduly affect the electrophoresis. Unfortunately, high concentrations of ionic species, such as guanidinium chloride, have a drastic effect on electrophoretic mobility and therefore cannot be included in electrophoretic gels. Small concentrations of other charged molecules making up the gradient are altered by their electrophoresis within the gel. Therefore reagents with a net charge are not ideal for TGGE, but any other agent that does not interfere with electrophoresis may be used in the transverse gradient.
For example, the size and shape of a molecule can be inferred from its electrophoretic mobility as a function of acrylamide and bisacrylamide concentrations (see Ferguson Plot). These electrophoretic measurements are usually carried out in a number of gels with different acrylamide concentrations, but they are more readily performed in a single transverse gradient gel in which the acrylamide concentration varies across the gel.
The most common agents used to alter the structures and conformations of proteins and nucleic acids are the denaturant urea and temperature. Their use in TGGE is described in the entries urea gradient electrophoresis and temperature gradient gel electrophoresis, where they give information about the folding and unfolding of proteins and nucleic acids.
