Gel Electrophoresis of Proteins and Nucleic Acids

Abstract

The electrophoresis technique describes migration of charged particles under the influence of an electric field. The rate of migration depends upon various factors like charge of the particle, applied electric field, and temperature and nature of the suspended medium. Gel electrophoresis is a technique in which the macromolecules like nucleic acids and proteins are forced to move through the pores of a gelatinous medium by applying an electrical current. The macromolecules are separated across the gel on the basis of size, electric charge, and other physical properties. The electrophoretic techniques involve the use of two types of gelatinous material, i.e., agarose gel and polyacrylamide gel. Agarose is usually used at concentrations between 0.7% and 3% which determines the pore size. Lower concentrations result in larger pore sizes, whereas higher concentrations result in smaller pore sizes. Agarose gels are generally used to separate larger nucleic acid molecules such as DNA or RNA because the pore sizes are large enough for these molecules to pass through the gel. DNA or RNA molecules having a net negative charge migrate toward the positive electrode (anode). The buffer serves as a conductor of electricity and controls the pH, which is important to the charge and stability of the biological molecules since DNA has negative charge at neutral pH and it migrates toward the positive electrode. Polyacrylamide gels are generally used for separation of small molecules like proteins. Pore size of the polyacrylamide gel varies according to its concentrations. The percentage of acrylamide in gels can vary from 3 to 30%. The low-percentage gels (e.g., 4%) having larger pore sizes are used in electrophoresis of proteins without any noticeable frictional effect. Two-dimensional gel electrophoresis (2D-PAGE) is a powerful and widely used method for the analysis of complex mixtures of proteins extracted from the cells, tissues, or other biological samples. Polyacrylamide gel elecrophoresis (PAGE) technique separates proteins according to two independent properties: the first dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pIs), and the second dimension is SDS-PAGE, which separates proteins according to their molecular weights (MWs). The isoelectric focusing involves placing the sample in gel with a pH gradient and applying a potential difference across it. In the electrical field, the protein migrates along the pH gradient, until it carries no overall charge. This location of the protein in the gel constitutes the apparent pI of the protein. The separation in the second dimension by molecular size is performed in slab SDS-PAGE. In this way, complex mixtures of different proteins can be resolved, and relative amount of protein can be determined.