Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis: Methods and Principles

Abstract

Electrophoresis is a technique used to separate and sometimes purify macromolecules—especially proteins and nucleic acids—that differ in size, charge, or conformation. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or gel. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. Agarose is typically used at concentrations of 0.5–2%. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000bp can be separated using standard electrophoretic techniques. SDS PAGE uses an anionic detergent (SDS) to denature proteins and the protein molecules become linearized. One SDS molecule binds to two amino acids. Due to this, the charge to mass ratio of all the denatured proteins in the mixture becomes constant. These protein molecules move in the gel (toward the anode) on the basis of their molecular weights only and are separated. The polyacrylamide chains are cross linked by N,N-methylene bisacrylamide comonomers. Polymerization is initiated by ammonium persulfate (radical source) and catalyzed by TEMED.