Nucleic acids electrophoretic mobility

Classical gel electrophoresis has been used extensively for protein and nucleic acid purification and characterization [9, 10], but has not been used routinely for small molecule separations, other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods, the analyte remains localized in the TLC bed and the mobile phase is immediately removed subsequent to chromatographic development. In contrast, in gel electrophoresis, the gel matrix serves primarily as an anti-... 

Gorin has extended this analysis to include (1) the effects of the finite size of the counterions in the double layer of spherical particles [137], and (2) the effects of geometry, i.e. for cylindrical particles [2]. The former is known as the Debye-Huckel-Henry-Gorin (DHHG) model. Stigter and coworkers [348,369-374] considered the electrophoretic mobility of polyelectrolytes with applications to the determination of the mobility of nucleic acids. 

In theory, if the net charge, q, on a molecule is known, it should be possible to measure / and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define /by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. 

Now, when the ionic front reaches the lower gel with pH 8 to 9 buffer, the glycinate concentration increases and anionic glycine and chloride carry most of the current. The protein or nucleic acid sample molecules, now in a narrow band, encounter both an increase in pH and a decrease in pore size. The increase in pH would, of course, tend to increase electrophoretic mobility, but the smaller pores decrease mobility. The relative rate of movement of anions in the lower gel is chloride > glycinate > protein or nucleic acid sample. The separation of sample components in the resolving gel occurs as described in an earlier section on gel electrophoresis. Each component has a unique charge/mass ratio and a discrete size and shape, which directly influence its mobility. 

There have been a number of refinements to the theory (Barkema el al., 1994 Duke et al., 1992 Lerman and Frisch, 1982 Levene and Zimm, 1989 Lumpkin et al., 1985, 1989), to include nucleic acid elasticity for example (Deutsch, 1988). There is no well-developed theory at the present time for nucleic acids containing helical junctions, although there have been recent attempts to incorporate this (Heuer et al., 2005 Saha et al., 2006). However, most electrophoretic data on junctions are analyzed empirically. The general rule that increased kinking results in lower mobility seems to work well, unless bending is so severe that helices clash (see, e.g., Goody et al., 2003). The very basis of the comparative approach is that the conclusions result from the interpretation of relative mobilities of matched species. 

Because conformational changes in RNA or short DNAs typically cause small changes in electrophoretic mobility, analysis of nucleic acid folding requires careful optimization of electrophoresis conditions. By contrast, protein—nucleic acid interactions are typically easier to analyze by native PAGE because the molecular weight and positive charge of the protein produces a relatively large shift in gel mobility. 

Affinity electrophoresis is an electrophoretic technique for the separation and characterization of proteins and nucleic acids on the basis of their affinity for the ligands that are immobilized within the separating matrix. Proteins which interact with immobilized ligands are retarded in their migration. This decrease in mobility is a function of the concentration of the immobilized ligands, and it reflects the strength of the interaction (Fig. 8.5). Thus, affinity... 

Separations by electrophoresis depend upon differences in rates of migration of the components in a mixture in an applied electric field. Provided the electric field is removed before ions in the sample mixture reach the electrodes, the components may be separated according to their electrophoretic mobility. Electrophoresis is thus an incomplete form of electrolysis. Electrophoresis is especially useful for analysis and separation of amino acids, peptides, proteins, nucleotides, nucleic acids and carbohydrates. 

The many hydroxyl groups on cellulose provide for extensive interaction between polar macromolecules and the paper electrophoresis strip. Therefore, hydrophilic proteins and nucleic acids tend to have low electrophoretic mobility. Acetylation of the cellulose hydroxyl groups produces a medium (cellulose acetate) that greatly speeds up electrophoreses. For cellulose acetate electrophoreses O.IM barbital buffer is the standard one. 

---