Gel electrophoresis is a fundamental laboratory technique used to separate molecules based on their movement through a porous matrix under an electric field. Yes, this method can be applied to proteins, but it requires specialized modifications. Unlike DNA, proteins are complex and diverse molecules that necessitate specific preparation to achieve meaningful separation. Protein electrophoresis methods are tailored to overcome these inherent molecular differences, making the technique a powerful tool in biochemistry and molecular biology.
The Structural Challenge of Protein Separation
Proteins present a significant challenge for simple gel electrophoresis because each molecule possesses a unique three-dimensional structure and a variable net electrical charge. The net charge is determined by the specific amino acid side chains and the surrounding pH of the buffer solution. Without modification, proteins separate based on an unpredictable combination of size, shape, and charge. This makes interpretation difficult, as the complex folded shape creates an inconsistent drag force. To effectively separate proteins, researchers needed a method that could standardize both their shape and their charge.
Separating Proteins by Molecular Weight: SDS-PAGE
The most common technique developed to address the structural challenge is Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). This method separates proteins solely based on their molecular weight or size. Standardization relies on the anionic detergent, Sodium Dodecyl Sulfate (SDS), incorporated into the sample preparation and the gel buffer.
SDS molecules bind to the protein backbone, causing denaturation—unfolding the protein into a linear, rod-like chain. This eliminates the native three-dimensional shape, standardizing the frictional drag. SDS also coats the protein chain with a uniform negative charge, masking the protein’s intrinsic charge. The ratio of SDS bound is proportional to the polypeptide chain length, ensuring the charge-to-mass ratio is nearly identical for every protein. With a uniform negative charge, all proteins migrate toward the positive electrode, governed primarily by size. Smaller proteins navigate the polyacrylamide gel (PAGE) matrix pores more easily and travel farther, resulting in separation by molecular weight.
Achieving Separation by Charge and Multi-Dimensionally
While SDS-PAGE separates by size, Isoelectric Focusing (IEF) separates proteins based on their intrinsic charge. IEF uses a gel containing a stable pH gradient. Proteins migrate under an electric field until they reach their isoelectric point (pI)—the specific pH where the protein carries no net electrical charge—and migration stops.
The highest resolution separation combines both methods into two-dimensional (2D) gel electrophoresis. This technique uses IEF in the first dimension, separating the protein mixture horizontally by charge (pI). The IEF gel strip is then placed on an SDS-PAGE gel, separating the proteins vertically by molecular weight in the second dimension. The result is a map of protein spots, each representing a distinct protein separated by both charge and size. This 2D approach can separate thousands of proteins simultaneously, making 2D gels a foundational technique in large-scale protein analysis, or proteomics.
Interpreting and Utilizing Protein Electrophoresis Results
After separation, proteins are made visible by staining the gel, revealing distinct bands (SDS-PAGE) or spots (2D gels). The location provides information on molecular weight, estimated by comparison to a ladder of known sizes, and, for IEF or 2D gels, the isoelectric point.
Comparing protein patterns between different samples, such as healthy versus diseased tissue, allows researchers to identify proteins expressed at different levels. This comparison can reveal potential biomarkers for disease or targets for drug development.
To confirm the identity of a specific protein, Western Blotting is often performed. This process transfers the separated proteins onto a membrane, where specific proteins are targeted and identified using antibodies. Western blotting utilizes the size separation achieved by electrophoresis to ensure the antibody detects the target protein at its correct molecular weight.