The solution of proteins to be analyzed is first mixed with SDS, a detergent which denatures the proteins and applies a negative charge to every protein. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in folding, as differences in folding patterns would cause some proteins to better fit through the gel matrix than others. Adding SDS solves this problem, as it denatures the proteins so that they may be separated strictly by length (number of amino acids). The SDS also binds to the protein in a constant ratio (1.4g SDS per 1.0g protein), giving a uniform mass:charge ratio to all proteins, so that the distance of migration through the gel is directly related to only the size of the protein. A stain is also added to the protein solution; this allows the experimentor to see the proteins at the end of the experiment.
The denatured proteins are then applied to one end of a layer of polyacrylamide gel submerged in water. An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty. After a set amount of time (usually a few hours), the proteins will have 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. Thus proteins may be separated roughly according to size (and therefore, molecular weight).
Because of the stain applied to all proteins, different proteins will form distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to determine the weight of unknown proteins by comparing the distance traveled relative to the marker.
Feeds |
Contact