Gel electrophoresis is a laboratory technique that separates biological molecules based on their physical properties. A specialized gel acts as a molecular filter, allowing scientists to analyze and isolate substances like DNA, RNA, and proteins. This method is fundamental for understanding genetic material and proteins, with broad applications in research, diagnostics, and forensics.
The Gel’s Composition
The gel used in electrophoresis is a porous, semi-solid matrix that provides a medium for molecular movement. Two primary types of gels are employed: agarose and polyacrylamide.
Agarose, derived from seaweed, forms a gel when heated in a buffer solution and then cooled. Its concentration determines the size of the pores, with lower concentrations yielding larger pores and higher concentrations resulting in smaller pores. This allows for the separation of larger molecules, such as DNA fragments ranging from approximately 100 base pairs to 25,000 base pairs.
Polyacrylamide gels are synthetic polymers formed through the chemical polymerization of acrylamide and a cross-linking agent, typically bis-acrylamide. The pore size is precisely controlled by adjusting the concentrations of acrylamide and the cross-linker during preparation. These gels create a finer, more uniform pore structure compared to agarose, making them suitable for separating smaller molecules like proteins (5 to 2,000 kilodaltons) and very small DNA fragments (under 500 base pairs).
How the Gel Separates Molecules
The gel functions as a molecular sieve, allowing molecules to move through its intricate network of pores under the influence of an electric field. Samples containing charged molecules are loaded into wells at one end of the gel, which is submerged in a buffer solution to conduct electricity and maintain pH. When an electric current is applied, negatively charged molecules, such as DNA and RNA due to their phosphate backbones, migrate towards the positive electrode. Proteins can also be separated this way, often by treating them to ensure a uniform negative charge.
As these charged molecules travel through the gel, their movement is impeded by the porous matrix. Smaller molecules navigate through the gel’s pores more easily and quickly, traveling further down the gel. Larger molecules encounter more resistance and move more slowly, remaining closer to the starting wells. This differential migration based primarily on size, and sometimes charge, results in the separation of molecules into distinct bands.
Unlocking Information: Visualizing Results and Applications
Once the separation process is complete, the molecules within the gel are typically invisible to the naked eye. To visualize the separated bands, the gel is usually stained with a specific dye that binds to the molecules of interest. For DNA, common stains like Ethidium Bromide fluoresce under ultraviolet light, revealing distinct bands that correspond to different DNA fragment sizes. Proteins can be visualized using stains such as Coomassie Blue or silver stain, which bind to the proteins and produce visible bands.
Visualizing these separated molecules provides valuable information for various applications:
- Forensics, for DNA fingerprinting in paternity testing or crime scene analysis.
- Research, to analyze gene expression, identify genetic mutations, and study protein purity and composition.
- Medical diagnostics, such as analyzing blood serum proteins to detect certain diseases.
- The food industry, to ensure product quality.
- Estimating the size of unknown molecules by comparing them to a ladder of molecules with known sizes.