Electrophoresis is a fundamental laboratory technique used to separate and analyze biological molecules based on their physical properties. The process involves placing a sample containing molecules, such as DNA or proteins, into a medium and applying an electric field. The molecules move through the medium at different rates, leading to their separation into distinct groups. This technique allows scientists to isolate and study components found in cells and tissues.
The Core Principle of Separation
The separation relies on the fact that most biological molecules carry an inherent electrical charge. DNA and RNA have a strong, uniform negative charge due to their phosphate backbone. Proteins, however, can have a positive or negative net charge depending on the surrounding environment’s pH.
When an electrical current is applied, charged molecules migrate toward the electrode with the opposite charge. Negatively charged molecules, such as DNA, move toward the positive electrode, known as the anode. The migration rate is directly influenced by the molecule’s net charge and size. Molecules with a higher charge and smaller size move faster through the medium.
The Essential Equipment and Setup
Electrophoresis requires a gel matrix, a buffer solution, and a power supply. The gel acts as a porous sieve through which the molecules travel, providing the physical basis for separation by size. Common gel materials include agarose, derived from seaweed, used for larger molecules like DNA, and polyacrylamide for smaller molecules like proteins.
The gel is submerged in a buffer solution that conducts the electric current and maintains a stable pH environment. This stable pH is necessary to preserve the molecules’ charge and structure. The power supply provides the direct current (DC) that creates the electric field, forcing the charged molecules to migrate.
To begin the process, the sample is loaded into small depressions, called wells, at one end of the gel. The gel is placed into a chamber, submerged in the buffer, and connected to the power supply. Applying voltage initiates the migration of the charged molecules through the gel’s pores.
Separating Biomolecules: How Samples are Run
The method for running a sample varies depending on whether the target molecules are nucleic acids or proteins. DNA separation, typically using agarose gel electrophoresis, separates fragments based on size. Since all DNA fragments have a nearly identical charge-to-mass ratio, smaller pieces encounter less resistance from the gel’s pores and travel faster and farther than larger pieces.
Protein separation often requires treating the samples with a detergent called sodium dodecyl sulfate (SDS) before loading them onto a polyacrylamide gel. SDS binds uniformly to the proteins, unfolding them and coating them with a consistently negative charge. This standardization ensures that the proteins separate primarily by mass, as their innate charge differences are masked.
After separation, the molecules are not visible, necessitating a visualization step. For DNA, a fluorescent dye that wedges itself into the molecule’s structure is often used. When the gel is exposed to ultraviolet light, the dye illuminates the separated groups, which appear as distinct “bands.” Researchers compare the position of these bands to a reference marker of known sizes, called a ladder, to accurately estimate the size of the molecules in the sample.
Real-World Uses of Electrophoresis
Electrophoresis has broad applications in medicine and forensics.
Genetic and Forensic Applications
In genetic analysis, it is used for DNA fingerprinting, comparing DNA patterns from different individuals. This is instrumental in paternity testing and criminal investigations. The separation of DNA fragments allows scientists to confirm the presence of specific genes or mutations associated with hereditary diseases.
Medical and Pharmaceutical Applications
In diagnostics, electrophoresis analyzes proteins in patient blood or urine samples. Serum protein electrophoresis helps identify abnormal protein levels or the presence of unusual antibodies, which can indicate diseases such as multiple myeloma or immune disorders. The technique is also used in pharmaceutical research to check the purity and concentration of therapeutic proteins and vaccines.