Electrophoresis is a technique in which charged molecules move through a supporting medium under the influence of an electric field, allowing separation of DNA, RNA or proteins based on their size and electrical charge.
Explanation
When an electric current is applied across a conductive medium, charged molecules migrate toward the oppositely charged electrode. Their rate of movement depends on the net charge, size and shape of the molecule as well as the properties of the medium. In gel electrophoresis, a matrix of agarose or polyacrylamide acts as a molecular sieve. Small molecules move more easily through the pores and travel farther than larger ones. Agarose gels with relatively large pores are used to separate nucleic acids ranging from a few hundred to tens of thousands of base pairs. Polyacrylamide gels with smaller pores are used for resolving proteins and small DNA fragments; when proteins are denatured with sodium dodecyl sulfate (SDS), the technique is called SDS‑PAGE and separates proteins primarily by mass. Isoelectric focusing separates proteins based on their isoelectric point within a pH gradient. Capillary electrophoresis uses narrow tubes and high voltages to achieve rapid, high‑resolution separations. Visualization of separated molecules is achieved with stains or fluorescent dyes and, for nucleic acids, by intercalating dyes such as ethidium bromide or SYBR Green. Electrophoresis is essential in molecular biology for checking polymerase chain reaction products, genotyping and DNA sequencing, and in clinical laboratories for analyzing serum proteins, hemoglobin variants and enzymes.
Types and Applications
Agarose gel electrophoresis is routinely used to size DNA fragments after restriction enzyme digestion or PCR amplification. SDS‑PAGE followed by transfer to a membrane forms the basis of Western blotting, which detects specific proteins using antibodies. Native PAGE preserves protein conformation and is used to study protein complexes. Capillary electrophoresis is employed in forensic analysis to separate short tandem repeats and in clinical chemistry analyzers. Two‑dimensional electrophoresis combines isoelectric focusing and SDS‑PAGE to resolve thousands of proteins from a cell lysate.
Electrophoresis exploits fundamental principles of physics to separate biomolecules, making it a cornerstone method in research and diagnostics.
Related Terms: Gel electrophoresis, DNA, Protein, SDS-PAGE, Isoelectric focusing