Electrophoresis is a laboratory technique that separates biological molecules based on their movement within an electric field. This process involves applying a voltage across a porous support medium, such as a gel, which contains the sample mixture. For proteins, the rate and direction of migration are determined by intrinsic physical properties that respond to the electrical force. Separation in a simple electric field is not based on a single, isolated factor.
The Primary Factors Governing Movement
When proteins are subjected to an electric current under standard, non-denaturing (native) conditions, their movement results from two fundamental properties: net electrical charge and molecular size and shape. The overall electrical charge dictates the direction and initial speed of migration. This net charge is highly dependent on the pH of the surrounding buffer solution because proteins are composed of amino acids with ionizable side chains.
If the buffer’s pH is higher than the protein’s isoelectric point (pI), the protein will carry a net negative charge and migrate toward the positive electrode. Conversely, a pH lower than the protein’s pI results in a net positive charge, driving movement toward the negative electrode. Simultaneously, the gel matrix acts as a molecular sieve, imposing a frictional resistance on the migrating molecules. Larger or more irregularly shaped proteins encounter greater resistance within the gel’s pores, which slows down their movement regardless of their charge. Therefore, under native conditions, separation is not solely by charge or by size, but by an inseparable combination of both factors.
Isolating Separation Based on Molecular Size
To separate proteins based on size, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is employed. This standardized technique overrides the influence of a protein’s native charge and shape, ensuring migration is determined almost exclusively by molecular weight. The process begins by treating the protein sample with the anionic detergent, Sodium Dodecyl Sulfate (SDS).
SDS denatures the protein, unfolding its complex three-dimensional structure into an extended, linear polypeptide chain. The detergent binds along the chain in a ratio proportional to the protein’s mass (about 1.4 grams of SDS per gram of protein). This binding masks the protein’s intrinsic electrical charge, coating it with a uniform, high-density negative charge. Since all proteins now possess a virtually identical negative charge-to-mass ratio, they migrate toward the positive electrode at speeds independent of their native charge.
Migration occurs through the polyacrylamide gel, which functions as a tightly controlled molecular sieve. Smaller protein chains navigate the meshwork of the gel pores with greater ease, allowing them to travel farther and faster. Larger proteins, however, are retarded by the sieving effect and move more slowly through the gel. The distance a protein travels becomes inversely proportional to the logarithm of its molecular weight. This relationship allows researchers to accurately estimate a protein’s mass by comparing its migration distance to a ladder of proteins with known molecular weights.
Isolating Separation Based on Net Charge
Isoelectric Focusing (IEF) is a specialized technique used to separate proteins based on their net charge, or more precisely, their isoelectric point (pI). The pI is the specific pH value at which a protein possesses an equal number of positive and negative charges, resulting in a net electrical charge of zero. Unlike uniform buffer systems, IEF utilizes a stable pH gradient established across the gel medium.
When a protein mixture is introduced to this gradient and an electric field is applied, each protein migrates according to the net charge it carries. A protein in an acidic region (low pH) is positively charged and moves toward the cathode (negative electrode). As it moves, it enters regions of increasing pH, gradually losing its positive charge. Conversely, a protein in a basic region (high pH) is negatively charged and moves toward the anode (positive electrode), entering regions of decreasing pH.
The protein continues to move until it reaches the precise point in the pH gradient where the surrounding buffer pH exactly matches its intrinsic pI. At this point, the protein has no net charge and ceases to respond to the electric field, becoming “focused” into a sharp, stationary band. Since each protein has a unique isoelectric point, IEF provides high-resolution separation based purely on subtle differences in charge properties, independent of size or shape.