Electrophoresis allows researchers to separate DNA and RNA, and proteins according to their size so as to identify, quantify and purify a specific fragment within the test sample. The application of an electric field across the sample-loaded gel (usually agarose or acrylamide) forces the negatively charged nucleic acid or protein to migrate towards the positive electrode. The gel is a porous matrix (acting as a sieve); allowing shorter fragments to travel quicker, and therefore further through the gel than longer fragments, due to less resistance, ultimately creating separation between different-sized fragments.
Basic Electrophoresis Technique
1) Cast gel if necessary and prepare running buffer
2) Fill the electrophoresis tank with running buffer so that the gel will be completely submerged
3) Add/insert gel
4) Mix the sample and standard (ladder) with loading dye and load a small volume of each into each well on the gel
5) Connect power supply electrodes to the appropriate ports
6) Turn on the voltage supply and set the current to the appropriate setting
7) Run the bands until they have separated as required
8) Turn off power and remove gel
An electrophoresis system usually compromises of a tank with chambers, casters, plates, spacers, combs and a power supply. They are set up to run either vertical (polyacrylamide) or horizontal (agarose) electrophoresis and provide the framework for running a gel.
Gels can be purchased precast or can be made up in the lab from a powder or solution. The two most common types of gel used for electrophoresis are agarose and acrylamide. All gels contain or must be created with equally-separated wells in which the sample can be loaded so that it can be pulled through the middle of the gel.
Agarose gels are used to separate DNA fragments. The pore size of agarose gels is not consistent; high concentration agarose gels are used to separate smaller DNA fragments and vice versa.
Acrylamide gels have a consistent pore size and are used for proteins ranging from 5-2000 kDa. Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) is a core step in western blotting. In this method proteins are mixed with SDS to denature them; unfolding them into a linear shape and applying a negative charge, both which are necessary for successful electrophoresis.
Molecular Standards, (DNA/RNA/Protein Ladders)
Standards (or ladders) contain several fragments of known lengths or molecular weights (MW). When ran on a gel this solution forms a series of bands which allow researchers to estimate the molecular weight of the test sample, (ran in parallel) and therefore purify the target molecule by its length or MW. The fragment can then be identified if it was previously unknown.
Loading Dyes are mixed with the sample before loading onto the gel; they increase the density of the nucleic acid or protein sample, thus sinking it into the well and preventing leakage, while also providing visual confirmation that the sample has migrated. The dye is also negatively charged so that it moves in the same direction as the nucleic acid or protein.
Stains , (e.g. fluorescent), help visualize the resulting bands after electrophoresis, under either visible or UV light. Ethidium Bromide, a UV stain, is commonly used for DNA and RNA.
Running buffer is required carry the current throughout the gel by providing ions while maintaining constant conditions such as pH. It also aids in keeping the gel cool, as the current running across the gel creates excess heat, which can cause agarose gels to melt.
The most common buffers used for agarose gels (and making up running buffers) are TBE (tris-borate-EDTA) and TAE (Tris-acetate-EDTA). TBE gives better resolution and therefore sharper bands, in particular for fragments larger than 1kb. However, TBE does make DNA extraction difficult. TAE is more popular than TBE, yet has a lower buffering capacity.
2D and 3D Electrophoresis
2D electrophoresis is used to separate a mixture of proteins by two properties; isoelectric point (pI) and molecular weight (MW), and therefore in two dimensions.
3D electrophoresis is a relatively new technology which separates proteins by native size, pI, and molecular mass.
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