SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) is a powerful technique which is used for the separation of proteins and nucleic acids. Electrophoresis is the migration of charged molecules in a media upon application of an electric field. The rate of migration depends on the charge on the molecule, its molecular mass, size and the strength of the electric field. Usually, this technique is routinely used for the analysis of proteins. The most commonly used matrix is agarose or polyacrylamide. These matrix forms a porous support and the size of the pores can be varied by changing the concentration of the matrix. Agarose is used mostly for separation of larger macromolecules, including nucleic acids, proteins and their complexes. On the other hand, polyacrylamide is used for the separation of proteins and small oligonucleotides. The charge on a protein is determined by the pH of the medium and the amino acid composition of the protein. Each protein has an isoelectric point which is the pH at which the protein has no net charge. Thus, at a pH below the isoelectric point, the protein will be net positive charge and migrate towards cathode, but at higher pH, it will be negatively charged and move towards anode. Thus, the movement of protein will not only depend on the mass, but also on the charge. Nucleic acids, however, remain negative at any pH due to the presence of the phosphate group of each nucleotide. Electrophoretic separation of nucleic acids is therefore strictly according to size. Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by binding to the polypeptide backbone. This makes the protein molecule negatively charged. This negative charge is proportionately distributed throughout the molecule, yielding the same charge density per unit length. In order to remove the disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size, the proteins are reduced either by 2-mercaptoethanol or dithiothreito. Thus, in denaturing SDS-PAGE separations, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. To increase the resolution of protein separation, a discontinuous buffer system is often used. The stacking gel contains a low pH, range of 6.8. At this pH, the major ion species, glycine, from the buffer is less ionized and hence moves very slowly. This leads to a trapping effect of the protein molecules, thereby concentrating them in the form of a band. As the protein enters the smaller pore sized separating gel and a higher pH, glycine is ionized, the voltage gradient is dissipated and the protein is separated based on size.