The denatured proteins are placed at one end of the polyacrylamide gel layer; submerged in a suitable buffer. Then, an electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Hereby, depending on their size, each protein will move in various distances through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty as they encounter more resistance. After, a few hours, the proteins can be measured, where it differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin.
Following electrophoresis, the gel can be stained (most commonly with Coomassie Brilliant Blue or silver stain), in order to visualize the separated proteins. After staining, different proteins will appear as distinct bands within the gel therefore, it is common to run molecular markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.
Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A co-migrating contaminant can appear as the same band as the desired protein. This co-migration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie Brilliant Blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification.
