The size and shape of biological molecules influences their migration rate. As a protein increases in size, its migration rate declines because of frictional forces in the electrophoretic buffer medium. Globular proteins exhibit a different migration rate from that of fibrous proteins. A molecule's charge depends on its dissociation at the pH of the buffer solution used. As a molecule's charge increases, its migration rate also increases.
The ions of the buffer used in the electrophoretic run conduct the current applied between the cathode and anode. The proteins in the sample interact with the ions in the buffer depending on the pH of the buffer. This interaction affects the migration rate of the protein molecules. Buffers with high ionic strengths cause an increase in current, which produces a greater amount of heat and reduction in the protein migration rate. Buffers with very low ionic strength lead to problems in protein diffusion; this impairs the separation of electrophoretic bands.
The migration of proteins depends on the characteristics of the electric field that is applied to the sample dispersed in the buffer. For a given voltage, the current that passes through the electrophoretic medium depends on the potential gradient and the medium's resistance. As the potential gradient increases, protein migration speeds up. As the length of the electrophoretic medium that proteins traverse increases, proteins encounter greater resistance. This hampers protein migration.
The time taken for separation of sample components is proportional to the voltage applied to the medium. An increase in voltage increases the migration velocity. However, high voltages generate an excessive amount of heat. Increased temperature alters the viscosity and electrical conductivity of the electrophoretic medium. This affects the migration rate as well. Temperature changes may also cause a change in the conformation of proteins, which reduces the migration velocity and efficiency of electrophoretic separation.