Definition
Gel electrophoresis is a laboratory technique used to separate macromolecules—typically nucleic acids (DNA or RNA) or proteins—based on their size, charge, and conformation by applying an electric field to a gel matrix.
Overview
In gel electrophoresis, a sample is loaded into wells of a gel composed of a polymeric matrix such as agarose (commonly used for nucleic acids) or polyacrylamide (commonly used for proteins). When an electric current is applied, charged molecules migrate through the gel: negatively charged nucleic acids move toward the anode, while proteins migrate according to their net charge at the buffer pH. The porous nature of the gel impedes movement, causing smaller molecules to travel faster than larger ones, resulting in size-based separation. After electrophoresis, separated bands are visualized using staining methods (e.g., ethidium bromide for DNA, Coomassie Brilliant Blue for proteins) or through fluorescence imaging.
Key applications include:
- Verification of DNA fragment size after polymerase chain reaction (PCR) or restriction enzyme digestion.
- Assessment of RNA integrity and size distribution.
- Analysis of protein expression, purity, and molecular weight.
- Preparation of samples for downstream techniques such as blotting or sequencing.
Variations of the basic method include:
- Denaturing gels (e.g., SDS‑PAGE for proteins, denaturing agarose gels for nucleic acids), which unfold biomolecules to separate solely by length or molecular weight.
- Native gels, preserving protein conformation and protein‑protein interactions.
- Two-dimensional gel electrophoresis (2‑D PAGE), which separates proteins first by isoelectric point and then by molecular weight.
Etymology / Origin
The term combines “gel,” referring to the semi‑solid polymer matrix that provides a sieving medium, and “electrophoresis,” derived from the Greek ‘ēlektron’ (amber, source of static electricity) and ‘phoresis’ (to carry or transport). The technique was first described in the 1930s for protein separation; agarose gel electrophoresis for nucleic acids became widespread in the 1970s following the development of agarose as a low‑melting-point gel.
Characteristics
| Feature | Typical Parameters |
|---|---|
| Gel matrix | Agarose (0.5–2 % w/v) for DNA/RNA; polyacrylamide (5–15 % w/v) for proteins |
| Buffer systems | TAE, TBE for nucleic acids; Tris‑glycine, Tris‑acetate for proteins |
| Voltage | 50–150 V for agarose gels; 100–200 V for polyacrylamide gels, often adjusted to maintain constant current |
| Run time | 30 min to several hours, depending on gel concentration and voltage |
| Visualization | UV transillumination (ethidium bromide, SYBR Gold), visible light imaging (Coomassie, silver stain), fluorescent labeling |
Related Topics
- Agarose gel electrophoresis – specific to nucleic acid separation using agarose matrices.
- Polyacrylamide gel electrophoresis (PAGE) – used primarily for protein and small nucleic acid fragments.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) – denaturing method that imparts uniform negative charge to proteins.
- Isoelectric focusing (IEF) – separates proteins based on their isoelectric point prior to PAGE in two‑dimensional electrophoresis.
- Western blotting, Southern blotting, Northern blotting – downstream techniques that transfer separated molecules from gels to membranes for hybridization or immunodetection.
- Capillary electrophoresis – a related technique employing thin capillaries instead of gels for high‑resolution separation.