Gel electrophoresis is an experimental procedure that uses an electric current to separate mixed proteins or nucleic acids based on their differing sizes, charges or structures. The procedure is used extensively in laboratory research in disciplines concerned with detecting DNA or analyzing microscopic particles, such as genetics, biochemistry, immunology and molecular biology. Gel electrophoresis is also a crucial first step in the protocol for other analytical procedures like Western Blotting (separation of proteins), Southern Blotting (DNA detection) and Northern Blotting (RNA detection).
How Gel Electrophoresis Works
Gel electrophoresis uses an electric current passed through a gel medium to separate the nucleic acids or proteins in a sample. As the sample migrates in the gel in response to the electric current, the small particles move more easily and more quickly than the larger particles, which results in a distinct banded pattern in the gel. This banded pattern is visualized via the application of staining agents, such as ethidium bromide, which reveals the gel bands under UV light, or silver stain, which is typically used to detect proteins. The silver stain is compatible with mass spectrometry techniques for further analysis of the protein composition.
Materials and Methods: Gels
The type of gel, percent concentration, and experimental protocol are highly dependent on the sample and purpose of the electrophoresis. Agarose and polyacrylamide are the most common types of gels used in electrophoresis. They can be prepared in variable concentrations depending on the type of sample being analyzed. For instance, higher concentration gels should be used to detect smaller proteins or nucleic acids. The gels can either be bought or prepared in the lab.
Agarose gels are simple to prepare, and have the added benefit of being non-toxic, as they are derived from seaweed. Polyacrylamide gels, however, must be prepared more carefully due to the potential toxic effects from acrylamide. The University of North Carolina provides a step-by-step guide to preparing an agarose gel, while the department of microbiology at the University of California Davis provides a simple protocol for preparing a polyacrylamide gel.
Loading Gel Samples
Samples are loaded into wells in the gel using a pipette. Because each sample is loaded into an independent lane, many different samples can be run at the same time without fear of contamination.
Running the Electrophoresis
The gel is immersed in an electrophoresis buffer to provide current-carrying ions and to maintain pH. The most common electrophoresis buffers are usually tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE). As in the selection of the gel, the type of buffer chosen also depends on the type of sample and purpose of the experiment. Samples are loaded into the gel with a loading buffer, which helps the samples to stay in the well and not disperse into the buffer solution. Tracking dyes may also be used to confirm that the samples are loaded correctly in the wells and to track the progress of the electrophoresis. As the electric current is applied, the particles move through the wells and form distinct separation bands on the gel.
Visualizing the Band Pattern
Using ethidium bromide, silver stain, radioactivity or another staining method the migration of the proteins or nucleic acids on the gel may be visualized and photographed. Composition of the sample may be further analyzed by performing PCR, mass spectrometry or blotting techniques.
Understanding Gel Electrophoresis
Gel electrophoresis may sound like a complex procedure, but it is a simple and effective way to separate proteins, purify DNA and prepare samples for further analysis. <© Copyright 2011 Erin Connelly, All rights Reserved. Written For: Decoded Science