Electrophoresis is an analytical method in which a controlled electric current is used in order to separate biomolecules according to their size to electric charge ratio, using a gelatinous matrix as a base. This technique has a variety of practical uses, such as forensic medicine for human identification, the human genome project, protein and genetic mutation research, and clinical diagnostic testing.
Electrophoresis is carried out with equipment composed of a negative charge at one end and a positive charge at the other, called an electrophoresis system. When inserting charged molecules, in this environment, the negative ones will go to one extreme and the positive ones to the other corresponding. For example, when analyzing proteins on a gel, in these kits, the entire protein is taken to analyze its size. In this way, the shorter ones will migrate to the poles more quickly and will be reflected in the lower part of the gel. Instead, the longest will be at the top.
The vertical gel electrophoresis technique works according to the primary theory of gel electrophoresis, but is considered to be more complex than the horizontal gel electrophoresis method. This technique uses a discontinuous buffer. A cathode is located in the upper chamber, and the anode is located in the lower chamber. The electrodes present in each compartment provide the required electric field. A thin layer of gel is poured between the two mounted glass plates.
Therefore, the upper part of the gel is immersed in the upper chamber, and the lower part of the gel is immersed in the lower chamber. Once the current is applied, a small portion of the buffer moves to the lower chamber from the upper chamber through the gel.
In vertical gel electrophoresis, the buffer only flows through the gel. This allows precise control of the voltage gradient during the separation stage. Acrylamide gel can be used as the compartments are not exposed to atmospheric oxygen. Due to the smaller pore size of the acrylamide gel, precise separation can be achieved with higher resolution.
Horizontal gel electrophoresis uses the basic theory for the separation of DNA, RNA or protein molecules according to their respective molecular size and charge. In this technique, the gel is present in a horizontal orientation and is immersed in a buffer that is continuous. The agarose gel is used to separate the gel box into two compartments.
One end of the gel box contains an anode, while the other end contains a cathode. When a current is applied, the buffer used in this technique allows the creation of a charge gradient. When the load is applied, the gel tends to heat up.
The buffer also functions as a coolant, keeping the temperature at optimal levels. Recirculation of the running buffer prevents the formation of a pH gradient. A discontinuous buffer system cannot be used in horizontal gel electrophoresis as the two compartments of the gel system connect with the running buffer.
Learn how gel electrophoresis separates DNA and protein fragments based on size and why one would use agarose gel electrophoresis versus SDS-PAGE.
In this technique, molecules are separated according to size and electrical charge. The gel used in gel electrophoresis is generally made from a material called agarose, which is a gelatinous substance extracted from seaweed. This porous gel could be used to separate macromolecules of many different sizes. The gel is immersed in a buffer solution of the salt in an electrophoresis chamber. Tris-borate-EDTA (TBE) is commonly used as the buffer. Its main function is to control the pH of the system. The chamber has two electrodes – one positive and one negative – at its two ends.
Agarose gel is a good electrophoretic support. In addition, its nature gives it the characteristic of being able to separate molecules based on their size. This support allows nucleic acids to be separated based on their size. It is used to separate large molecules.
Polyacrylamide gel is considered the best support. They also have great mechanical stability and are insoluble in water. Among so many advantages there is a drawback that is limiting its use: it is neurotoxic. It is used for the separation of proteins and, like the agarose gel, allows the separation of molecules based on their size.
This technique differs a lot from the previous ones. The support is different, since it uses fused silica capillaries, covered with a layer of polyimine. The results are visualized through a computer screen.
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Electrophoresis is an analytical method in which a controlled electric current is used in order to separate biomolecules according to their size to electric charge ratio, using a gelatinous matrix as a base..
Gel electrophoresis is a laboratory technique used in genetics to separate mixtures containing DNA, RNA, and other proteins according to their respective molecular size and charge...