Electrophoresis is the process whereby ions move through a medium in response to an applied electric field. Separation of ions by electrophoresis exploits the fact that the rate of motion of charged particles in any particular applied electric field is directly proportional to their charge and  inversely proportional to their size and the viscosity of the medium. For these reasons, small and highly charged molecules will move a greater distance than will large and lower charged molecules in the same period of time. The efficiency of electrophoretic separation of ions is thus determined by the relative charge densities (charge per unit volume) of the ions in the mixture. In gel electrophoresis the medium is typically polyacrylamide or agarose, a viscous media that is required to minimize diffusion of the constituent ions.

The two most commonly used forms of protein electrophoresis are sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). In SDS-PAGE, protein samples are first denatured by heating in SDS. Denaturation in SDS results in the coating of the polypeptides with negatively charged SDS molecules, rendering each protein highly negatively charged. The denatured proteins are then applied to one end of a slab of polyacrylamide. Electrodes are then attached to opposite ends of the slab with the cathode (-) at the end where the denatured protein was applied and the anode (+) to the other end. Application of voltage across the gel forces the negatively charged proteins to migrate towards the anode. In the highly viscous polyacrylamide, larger denatured proteins will experience greater frictional drag than smaller denatured proteins and move through the gel matrix at a much slower rate. After a predetermined period of time, the electrodes are removed and the gel slab is stained with dye to show the locations of proteins in the gel. The smaller proteins are found closer to the anode than the larger ones. An inverse log-linear relationship of protein molecular weight to the distance traveled is typically observed.

In contrast to the molecular weight separation achieved by SDS-PAGE, IEF separates proteins by their isoelectric point, the pH at which they are uncharged. Since most proteins contain both acidic and basic amino acids, the overall charge of a protein will change from highly positive at low pH to highly negative at high pH. At some pH, determined by the number of acidic and basic side chains present in the protein, the protein will be uncharged. In a typical IEF experiment, a protein mixture  is applied to the center of a polyacrylamide gel slab. Alternate ends of the gel are dipped in either an acidic (anode) or basic solution (cathode) respectively. Voltage is then applied across the gel though these solutions. Immediately, protons begin to migrate toward the cathode and hydroxide ions toward the anode. Within minutes these migrating ions establish a linear pH gradient across the gel. To maintain the gradient, special buffers known as ampholytes are added to the gel before hand. Depending on the particular protein's pI it will be either positively or negatively charged after the gradient is established and will then slowly migrate toward either the cathode or anode respectively until it reaches the point on the gel where it is no longer is charged (pH =pI) and ceases to migrate further.

A third permutation of gel electrophoresis is known as two-dimensional gel electrophoresis (2D-PAGE or 2D gels). Here, proteins are first separated by IEF and then orthogonally by SDS-PAGE. A detailed presentation of this method can be found on our 2D Gel electrophoresis page.

Proper sample preparation is paramount for successful execution of protein gel electrophoresis. An introduction to sample preparation is provided on our Sample Preparation page. Proper sample fractionation is a precursor to further analysis using mass spectrometry. Protein gel electrophoretic methods provide one path to facilitate the required fractionation. Methodologies employed for the isolation of proteins from gels and subsequent processing for mass spectrometric analysis are dependent on the source of the sample, the desired end result and nature of the target protein.