Explanation:
Electrophoresis depends on the ability of charged molecules to migrate when placed in an electric field. The electrophoretic separation of proteins is usually accomplished using polyacrylamide gel electrophoresis (PAGE), in which the proteins are driven by an applied current through a gelated matrix.
A polyacrylamide gel may be formed as a thin slab between two glass plates or as a cylinder within a glass tube.
Step 1:
Once the gel has polymerized, the slab (or tube) is suspended between two compartments containing buffer in which opposing electrodes are immersed.
Step 2:
In a slab gel, the concentrated, protein-containing sample is layered in slots along the top of the gel. The protein sample is prepared in a solution containing sucrose or glycerol, whose density prevents the sample from mixing with the buffer in the upper compartment.
Step 3:
A voltage is then applied between the buffer compartments, and current flows across the slab, causing the proteins to move toward the oppositely charged electrode.
(Separations are typically carried out using alkaline buffers, which make the proteins negatively charged and cause them to migrate toward the positively charged anode at the opposite end of the gel).
Step 4:
Following electrophoresis, the slab is removed from the glass plates and stained.
The relative movement of proteins through a polyacrylamide gel depends on the charge density (charge per unit of mass) of the molecules. The greater the charge density, the more forcefully the protein is driven through the gel, and thus the more rapid its rate of migration.
Step 5:
The progress of electrophoresis is followed by watching the migration of a charged tracking dye that moves just ahead of the fastest proteins.
After the tracking dye has moved to the desired location, the current is turned off, and the gel is removed from its container.
Along with charge density size and shape also play a role in PAGE fractionation.
Polyacrylamide forms a cross-linked molecular sieve that entangles proteins passing through the gel. The larger the protein, the more it becomes entangled, and the slower it migrates.
The concentration of acrylamide (and cross-linking agent) used in making the gel is also an important factor. The lower the concentration of acrylamide, the less the gel becomes cross-linked, and the more rapidly a given protein molecule migrates.
