Proteins perform nearly all functions within a cell, and scientists often need to separate them from complex mixtures to study them. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, widely known as SDS-PAGE, is a laboratory technique designed to achieve this separation. The goal of this method is to isolate proteins based exclusively on their molecular weight, or size. This procedure works by applying an electric current to drive proteins through a specialized physical barrier, or gel, that allows smaller molecules to move faster than larger ones.
Preparing Proteins for Size-Based Separation
Proteins in their natural state have complex shapes and varying electrical charges, which interfere with separation based solely on size. To eliminate these factors, samples are treated with Sodium Dodecyl Sulfate (SDS). SDS is an anionic detergent that acts to completely unwind, or denature, the proteins, breaking down their complex structures into simple linear chains.
This unwinding ensures all proteins assume a rod-like shape, preventing movement from being hindered by unique folding patterns. As the proteins unfold, SDS molecules bind along the entire length of the polypeptide chain in a consistent ratio. This uniform coating provides a large, negative electrical charge that overwhelms the protein’s native charge.
This preparation converts all proteins into linear strands possessing a nearly identical negative charge-to-mass ratio, regardless of their original charge or complex shape. Because the charge is masked and the shape is standardized, the only variable determining protein behavior in an electric field is its length, which corresponds directly to its molecular mass.
The Polyacrylamide Gel as a Molecular Sieve
The prepared proteins are loaded into the polyacrylamide gel, which functions as a molecular sieve. The gel is synthesized through the polymerization of acrylamide monomers and a cross-linker, creating a highly porous, mesh-like structure. This meshwork provides the physical resistance necessary for molecular separation.
The size of these pores is precisely controlled by the concentration of the acrylamide used in the gel’s formation. A higher percentage of acrylamide creates a tighter mesh with smaller pores, which is ideal for resolving smaller proteins. Conversely, a lower percentage gel has larger pores, allowing larger proteins to be separated more effectively.
Tailoring the pore size allows researchers to select a gel that provides optimal separation for the specific range of protein sizes being analyzed. The polyacrylamide matrix itself remains chemically inert throughout the process, providing a stable, high-friction environment that physically impedes the passage of molecules.
Differential Migration and Separation Under Current
Separation begins when the polyacrylamide gel is placed in a buffer solution and an electrical current is applied. Since the SDS-coated proteins possess a strong net negative charge, they are repelled by the negative electrode (cathode) and pulled toward the positive electrode (anode). This movement of charged molecules in an electric field is electrophoresis.
Because the proteins have a standardized charge-to-mass ratio, the force pulling them through the gel is virtually the same for every molecule. As the proteins navigate the microscopic pores of the polyacrylamide mesh, they encounter physical resistance, which differentiates their speed. Larger, longer SDS-protein complexes experience significantly more friction and obstruction as they squeeze through the small openings.
Smaller proteins, being shorter and more compact, can slip through the pores of the molecular sieve more easily and encounter less hindrance. Consequently, smaller proteins travel faster and migrate further down the gel toward the positive electrode. Larger proteins are retarded, remaining closer to the loading point. This differential rate of movement causes the protein mixture to separate into distinct bands, with the smallest proteins at the bottom and the largest near the top.