polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. At the pH at which gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresi An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel away from the negative electrode (which is the cathode being that this is an electrolytic rather than galvanic cell) and towards the positive electrode (the anode). Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty. The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. The gel mobility is defined as the rate of migration traveled with a voltage gradient of 1V/cm and has units of cm2/sec/V. For analytical purposes, the relative mobility of biomolecules, Rf, the ratio of the distance the molecule traveled on the gel to the total travel distance of a tracking dye is p Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids. In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains. When subjected to an electric field in PAGE, the negatively charged polypeptide chains travel toward the anode with different mobility. Their mobility, or the distance traveled by molecules, is inversely proportional to the logarithm of their molecular weight. By comparing the relative ratio of the distance traveled by each protein to the length of the gel (Rf) one can make conclusions about the relative molecular weight of the proteins, where the length of the gel is determined by the distance traveled by a small molecule like a tracking dye. For nucleic acids, urea is the most commonly used denaturant. Proteins that have a greater hydrophobic content – for instance, many membrane proteins, and those that interact with surfactants in their native The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. The gel mobility is defined as the rate of migration traveled with a voltage gradient of 1V/cm and has units of cm2/sec/V. For analytical purposes, the relative mobility of biomolecules, Rf, the ratio of the distance the molecule traveled on the gel to the total travel distance of a tracking dye is plotted versus the molecular weight of the molecule (or sometimes the log of MW, or rather the Mr, molecular radius). Such typically linear plots represent the standard markers or calibration curves that are widely used for the quantitative estimation of a variety of biomolecular sizes. Certain glycoproteins, however, behave anomalously on SDS gels. It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to the marker. For proteins, SDS-PAGE is usually the first choice as an assay of purity due to its reliability and ease. The presence of SDS and the denaturing step make proteins separate, approximately based on size, but aberrant migration of some proteins may occur. Different proteins may also stain differently, which interferes with quantification by staining. PAGE may also be used as a preparative technique for the purification of proteins. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass-charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide. Urea (CO(NH2)2; mW: 60.06) is a chaotropic agent that increases the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds and van der Waals forces.