Electrophoresis

Electrophoresis Introduction Electrophoresis is a separations technique that is based on the the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.For safety reasons one electrode is usually at ground and the other is biased positively or negatively. Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

Instrumentation An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the support is stained to visualize the separated components. Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At this pH the protein is netural and no longer migrates, i.e, it is focused into a sharp band on the gel. Schematic of zone electrophoresis apparatus

Discontinous Electrophoresis

Introduction Discontinous electrophoresis uses two gels that are buffered at different pHs. When proteins migrate from one gel to the other they become concentrated into sharp bands, which produce higher resolution than in conventional electrophoresis. Schematic of discontinous electrophoresis technique

Capillary Electrophoresis (CE) Introduction Performing electrophoresis in small-diameter capillaries allows the use of very high electric fields because the small capillaries efficientlydissipate the heat that is produced. Increasing the electric fields produces very efficient separations and reduces separation times.

Instrumentation Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length. The applied potential is 20 to 30 kV. Due to electroosmotic flow, all sample components migrate towards the negative electrode. A small volume of sample (10 nL) is injected at the positive end of the capillary and the separated components are detected near the negative end of the capillary. CE detection is similar to detectors in HPLC, and include absorbance, fluorescence, electrochemical, and mass spectrometry.

The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection. Schematic of capillary electrophoresis

Electroosmotic flow The surface of the silicate glass capillary contains negatively-charged functional groups that attract positively-charged counterions. The positively-charged ions migrate towards the negative electrode and carry solvent molecules in the same direction. This overall solvent movement is called electroosmotic flow. During a separation, uncharged molecules move at the same velocity as the electroosmotic flow (with very little separation). Positively-charged ions move faster and negatively-charged ions move slower. Schematic of the double layer on the capillary surface

SDS-PAGE Introduction SDS-PAGE stands for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and is useful for molecular weight analysis of proteins. SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight.