Objective

To separate protein based on their charge and size.

Theory

Polyacrylamide Gel Electrophoresis (PAGE) is an ideal analytical method used for protein and relatively small nucleic acid molecules separation and analysis. This method separate components of a protein mixture based on their both charged and size, charged molecules will migrate in an electric field towards positively charged electrode (anode) by sieving effect. As in general gel electrophoresis, the molecule will run in their native state, preserving the molecules’ higher-order structure, molecular weight of biological molecules cannot be determined because the mobility of a substance in the gel depends on both size and number of electric charged they carry. Therefore, the protein molecules need to be denatured with an appropriate denaturing agent. Urea is the commonly used to denature nucleic acid. Sodium dodecyl Sulfate (SDS) is an anionic detergent used to unfold protein molecules to linearize proteins (proteins will lost their secondary, tertiary and quaternary structure) and impart them with extra negative charge. SDS biding to the polypeptide chain provide them with even distribution of charge per unit mass, thus resulting in a fractionation by approximate size during gel electrophoresis process. This method is called SDS-PAGE. The other PAGE methods such as isoelectric focusing and 2D PAGE also can be applied for the separation of proteins molecules based on their distinct molecular properties. All subunits can be visualized by various staining (protein-specific) technique, the relative amounts of these proteins (subunits) can also be determined and the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder (marker).