Gel electrophoresis refers to using a gel as an anticonvective medium and or sieving medium during electrophoresis. Gel electrophresis is most commonly used for separation of biological macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein; however, gel electrophoresis can be used for separation of nanoparticles. Electrophoresis refers to the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
Types of ElectrophoresisElectrophoresis is one of the most popular ways to analyze macromolecules such as proteins, DNA and RNA. There are several types of electrophoresis, but the concepts are similar. The machine has an anode (positive charge) and a cathode (negative charge). Negative ions move toward the anode, and positive-charged ions move towards the cathode. The rate and distance traveled by these molecules help scientists classify and study different biomolecules.
SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) is a common form of electrophoresis for analyzing proteins. The SDS part of the name is a protein denaturing detergent that causes the molecule to unfold. The detergent binds to the polypeptide in a 1:1 ratio with each segment of the protein to give it a charge. The protein polypeptides move through the gel at different rates depending on mass, allowing researchers to study proteins based on size.
Agarose gels are an electrophoresis method to separate RNA and DNA molecules. Like SDS-PAGE,this separates the molecules based on charge and size. DNA molecules are negatively charged, so they move through the gel quickly depending on size. Smaller DNA fragments move more quickly than larger ones due to friction resistance.
Electrofocusing takes advantage of charge and pH values of proteins. A container is filled with a gel solution that has an increasing pH gradient. The amino acids that form polypeptides have different acidic or basic charges. The protein travels through the gel, obtaining or losing protons depending on its charge. As the protein particle moves through the gel, it eventually becomes neutral and gets stuck in an isoelectric position.
Capillary electrophoresis is a method similar to SDS-PAGE. It separates molecules based on their charge and mass. Molecules are placed in rows called capillaries filled with conductive, electrolyte fluid. The analytes move in a speed relative to their charge and mass. This method is an older technique introduced in the 1960s. SDS-PAGE is usually preferred in labs.
Native gels are similar to SDS-PAGE, except the detergent (SDS) is not used to denature proteins. Native gels are only able to separate proteins up to 2,000 kDa in size. Because the proteins are left folded, the dyes used are also different than SDS-PAGE. Researchers are able to view proteins based on mass and charge using enzymes that catalyze protein-specific reactions.
Principles of Electrophoresis