Electrophoresis is a process that separates charged particles in a fluid with the aid of the field of electrical charge. In life sciences, an electrophoresis is a vital tool that separates protein molecules or DNA according to its size and type.
In a laboratory setting, electrophoresis is used to separate molecules according to heir size, purity, and density.
Image 1: The image above shows how an agarose gel electrophoresis is done.
Picture Source: addgene.org
There are different types of electrophoresis and the most common types are as follows:
It is probably the simplest type of electrophoresis wherein the sample is applied on a strip of filter paper moisturized with a buffer solution.
The end of the strip is dipped into separate tanks that have a buffer solution and different electrodes. A current is applied which will make the sample move towards the electrode with opposite polarity. The strip is dried and checked under the detection system.
Agarose gel electrophoresis
It uses an agarose gel to separate fragments of DNA or RNA of varying lengths. It observes the movement of negatively charged RNA or DNA molecules from the negative electrode to the positive ones. The molecules are differentiated according to the size of molecules.
Polyacrylamide gel electrophoresis
there are two types of gels used – dissociating and non-dissociating. The non-dissociating gel separates proteins in the original form thereby conserving the structure, function, and activity of the protein. On the other hand, the dissociating gel denatures protein into polypeptides to find out the compositionof a given sample. (1, 2, 3, and 4)
Image 2: The image above described how DGGE works.
Picture Source: pinimg.com
Denaturing gradient gel electrophoresis
also known as DGGE and temperature gradient gel electrophoresis – The former is used to separate PCA generated DNA products, which is vital in molecular fingerprinting. It is distinct from the rest because it separates PCR products according to its size difference and denaturing rate.
On the other hand, temperature gradient gel electrophoresis functions in a way like the denaturing gradient gel electrophoresis. However, its separating function is dependent on the temperature gradient. The temperature gradient enables the sample to denture based on the site mutations.
Through its use, researches can identify a single base pair mutation in a given sample (heterogeneous sample) as the denaturing point could either be lower or higher from the sample. (5, 6, 7, and 8)
Image 3: An image representation of two-dimensional electrophoresis.
Picture Source: wikimedia.org
Image 4: The principle of pulse-field gel electrophoresis as shown in the image above.
Picture Source: ytimg.com
Isoelectric focusing and 2-D gel electrophoresis
The former is a method of protein separation according to net charge. A sample of protein is placed in a pH gradient slab generated by an electrical field. It resulted in the migration of protein in the pH gradient field until such time it reaches a pH with isoelectric point of zero.
On the other hand, the latter, 2-D gel electrophoresis combines SDS-PAGE and isoelectric focusing method thereby separating proteins according to their size and isoelectric point. The 2-D gel electrophoresis is the most preferred method of the two because it gives a better protein resolution. It is also useful in separating protein if the protein’s size and charge are unknown.
It is a method of electrophoresis that enables enzymatic activity to be checked in situ after an enzyme underwent electrophoresis. Through this method, researchers can characterize proteins/enzymes present on the gel without the need to purify the protein or enzyme.
Through this process, the researcher will be able to save time by not undergoing anymore the enzyme or protein purification step. Zymograms enables you to check the physical characteristics of the enzymes such as the isoelectric point and molecular weight. (3, 6, and 9)
The usual electrophoresis method is not applicable in very large DNA molecules, typically around 30kb to 50kb. However, pulse-field electrophoresis can withstand the examination of even large DNA molecules making it the most suitable type of electrophoresis.
It uses a thin capillary tube made of either plastic, quartz, or glass. The tube is filled with the required buffer for agarose work. Capillary electrophoresis is used for analytical work such as genetic analysis, counter-ion analysis, pharmaceuticals with enantiomers, and characterization of protein.
It is a more efficient type of electrophoresis because it leads to an increase in throughput by many folds over the capillary electrophoresis system because microchip system has various micro-channels enabling high throughput experiments to be done in the easiest and the most efficient way possible.
This type of electrophoresis is fully automated from the very start (handling of sample) to the end (data analysis). Since it is fully automated, the chances of human error can be reduced significantly.
Fluorophore-assisted carbohydrate electrophoresis
It helps in identifying carbohydrates with the attached fluorescent dye by means of carbohydrate separation with the use of polyacrylamide gel.
It is useful in analyzing various types of carbohydrates like glycolipids, glycoproteins, and polysaccharides both plants and bacteria.
This type of electrophoresis simplifies the process of detecting lipid-linked oligosaccharides, which might be needed in a sample to be labeled with radioactive sugar precursors before the molecules are detected.
It is a type of electrophoresis wherein the resolving capability of the capillary electrophoresis is used to differentiate samples that undergo interactions of both specific and non-specific during electrophoresis.
What makes it special is its ability to detect affinity interactions be it in free or immobilized form. It can detect peptides and proteins. It is also vital in detecting small molecules, development of drugs, and for immune-affinity activities.
The sensitivity of affinity electrophoresis enables a more precise detection and discrimination of proteins, both normal and carcinogenic in a given sample.
Automated electrophoresis system
electrophoresis can now be done with the use of a computerized robotics and programming. It allows electrophoresis protocols to be automatically conducted. (2, 5, 9, and 10)
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- Agarose gel electrophoresis of DNA – Principle, Protocol and Uses
- Capillary Electrophoresis – Applications and Procedure
- Southern Blot : Principle, Protocol (steps) and Uses
- Immunoelectrophoresis Test – Principle (Steps), Uses, Limitations and Facts