Electrophoresis


Electrophoresis


General description


Electrophoresis (from the greek: phoresis=migration) refers to the phenomenon where dispersed particles under the influence of a uniform electrical field experience motion as a result of that electrical field[1][5].
The dispersed particles have an electric surface charge, on which the electrical field exerts an
electrostatic force. Thus the particle will migrate due to the effect of the electrical field. The direction of migration is defined by the electrical surface charge of the dispersed particles. If it is globally positive it will migrate to the direction of the negative electrode. If it is globally negative then the particle will migrate to the positive electrode. In opposition to the electrostatic force, there are two other forces that have an opposing effect on the migration of the dispersed particles. These are the friction force and the electrophoretic retardation force. The second one is explained by the double layer theory[1]. According to this theory when a charged surface is in contact with a fluid, the electrostatic forces stimulate the formation of a layer around the surface with an opposing charge. The Debye length is the distance over which significant charge separation can occur in this layer[11]. The electrical field influences this layer, and sin

ce it has an opposing electrical charge to the surface suffers a force in the opposite direction of that of the particle thus slowing the migration[1].
Several mathematical models were developed to predict variables concerning the electrophoresis, such as the electrophoretic mobility, normally designed by µe. In the case of a low Reynolds number and moderate electrical field strength, this variable is defined as:


fisrt_formula.png
Field_strengh.png
Representation of the forces in operation during an electrophoreses. From http://en.wikipedia.org/wiki/File:Electrophoresis.gif
[1]


where v is the velocity of a dispersed particle and E is electrical field strength. One of the first formula to appear and that is still widely used nowadays is the Smoluchowsk formula:

second.png[1]

In this formula εr is the dielectric constant of the dispersion medium, ε0 is the permittivity of free space, η is dynamic viscosity of the dispersion medium, and ζ is zeta potential, which is the electric potential in the interfacial double layer. The reason why this formula is so widely used is because of its simplicity and because it works for particles with any shape and any concentration. Though this simplicity is in itself a problem because this formula does not include the Debye length, nor it includes the surface conductivity, which is the additional conductivity that a fluid has near a solid surface. The Smoluchowsk formula is only able to give accurate results when the double layer is thin and the surface conductivity is insignificant[1]. For cases when the double layer is thick, another formula has been developed by Erich Huckel.
third.png[1]
There are also more complex analytical theories which are able to eliminate the restrictions of a small surface conductivity, pioneered by Overbeek and Booth, but they are not frequently used due to their complexity[1].
Even though this method was developed for use in charged molecules only, recent studies in the area of molecular dynamics seem to indicate that even neutral particles can migrate under the influence of an electrical field. In this case, electrophoresis is possible due to the movement of the water molecules near the particles. The water molecular structure is affected by the electrical field and the particle is dragged along with the water[1].

Pratical Uses


The electrophoresis is used to separate molecules. Its simplicity and the fact that there are a multiple types of electrophoresis capable of separating several types of molecules and capable of separating the same type of molecules based on physical differences. For this purpose, many different types of electrophoresis were developed. The most common ones will be listened and a small description will be provided.
  • Affinity electrophoresis
  • Capillary electrophoresis
  • Electroblotting
  • Electrophoretic display
  • Electrophoretic light scattering
  • Electrophoretogram
  • Gel electrophoretic
  • isotachophoresis
  • SDS-PAGE
  • 2-D electrophoresis

SDS.png
Representation of the SDS-PAGE electrophoresis. From http://www.molecularstation.com/pt/sds-page-gel-electrophoresis/


SDS-PAGE


The SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used to separate proteins in a gel that acts as a physical support. It is widely used in biochemistry, forensic and molecular biology and it separates the proteins according to their electrophoretic mobility. The sodium dodecyl sulfate (SDS) binds to the protein and, since it has a negative charge, it gives all proteins a linear structure, and the same charge per unit mass, so the result is the fractionalization by size. Then the proteins are applied in the negative electrode and the electrophoresis is activated. Since all proteins are negative, due to the SDS, they will all migrate towards the positive electrode. The smaller proteins will migrate faster because of their lower mass[2][7].




DNA.png
(A)-Representation of an DNA electrophoresis. (B)-A image of a developed gel of an DNA electrophoresis. (C)-Representation of the development of the gel. From http://www.bio.miami.edu/~cmallery/255/255tech/ecb10x5b.jpg



Gel Electrophoresis


This is a technique used to separate deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules. The reason why it is called gel electrophoresis is due to the gel used as a support where the samples are applied. This technique is more normally applied to DNA or RNA. For protein molecules, it is normally used the SDS-PAGE. This technique is normally used for analytical purposes, and it is often coupled with other analytical techniques, such as mass spectrometry, PCR, among others. For this technique, the DNA or RNA must be in linear form, so that the migration has a relation with the molecule size. Normally, to guarantee this linearity, the DNA and RNA is treated with an endonuclease. The gel can be manipulated in order to obtain different results. The porosity of the gel can be adjusted so that it only separates a small range of sizes, for example[5].




Isotachoelectrophoresis

The isotachophoresis (from the greek: iso=equal; tachos=speed; phoresis=migration) uses different types of electrolyte, which is the solution that carries the current from one electrode to another. A fast electrolyte is used on the negative electrode a
2-D.png
A representation of a two-dimension electrophoresis. From http://www.bio.miami.edu/~cmallery/255/255tech/mcb3.33a.2D.jpg
nd a slow electrolyte is used on the positive electrode. This causes the appearance of an electrical field that is not uniform. The electrical field is stronger on the first electrolyte and weaker on the last one. Then the sample is applied between the fast initiating electrolyte and the slow terminating electrolyte. The molecules on the sample are then separated according to their electrophoretic mobility. The molecules will concentrate on bands in places which correspond to their electrophoretic mobility. To use this technique the chosen electrolytes must have a lower and a higher electrophoretic mobility[3].

Two-dimensional electrophoresis

The two-dimensional gel electrophoresis (2-DE) is a gel based technique where in fact two electrophoresis are done in a single gel. Unlike normal electrophoresis where samples are applied along a line, in 2-DE a single sample is put in a corner of the gel and a first electrophoresis is done in one direction. Then, when it is over, a second electrophoresis is done in a perpendicular direction of the first one. This way the sample is separated in two directions, and by applying different electrophoresis, is possible to separate the molecules according to two different characteristics, such as size and isoelectric point[4].


History


1807 - Reuss describes the phenomena where charged dispersed particles in solution migrate under the influence of an uniform electrical field[1]

1903 - Marian Smoluchowsk developed the first formula used to calculate the electrophoretic mobility[1]

1952 - The paper electrophoresis is established[12]

1957 - The thin-layer electrophoresis on silica, cellulose and polyamide is established by Kohn[12]

1960 - The polyacrylamide gel electrophoresis (PAGE) is established by Samuel Raymond[10]

1967 - Shapiro uses the sodium dodecyl sulfate in the PAGE, thus establishing the SDS-PAGE[10]

1975 - The isotachophoresis is established[12]

1988 - The 2-D electrophoresis is established[12]


Prepared by Daniel Filipe Camarneiro Silva

1. http://en.wikipedia.org/wiki/Electrophoresis
2. http://en.wikipedia.org/wiki/SDS_PAGE
3. http://www.aesociety.org/areas/isotachophoresis.php
4. http://www.wzw.tum.de/blm/deg/manual/manualwork2html02test.htm
5. http://www.molecularstation.com/pt/dna/dna-gel-electrophoresis/
6. http://en.wikipedia.org/wiki/File:Electrophoresis.gif
7. http://www.molecularstation.com/pt/sds-page-gel-electrophoresis/
8. http://www.bio.miami.edu/~cmallery/255/255tech/ecb10x5b.jpg
9. http://www.bio.miami.edu/~cmallery/255/255tech/mcb3.33a.2D.jpg
10. http://bbwiki.tamu.edu/index.php?title=SDS-page
11. http://en.wikipedia.org/wiki/Electrical_double_layer
12. http://people.inf.elte.hu/nadnabt/Muszeres_Anal/Kromatografia/KapilElektroforezis.pdf

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