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REVIEW ON ELECTROPHORESIS TECHNIQUES

About Author:
Kambham Venkateswarlu
Final Year Graduate student
Sri Lakshmi Narasimha College of Pharmacy,
Palluru, Chittoor-517132, Andhra Pradesh, India
k.v.reddy9441701016@gmail.com

ABSTRACT:
Electrophoresis is also called as cataphoresis. It is the motion of dispersed particles relative to a fluid under the influence of spatially uniform electric field. This electro kinetic phenomenon was observed for the first time in 1807 by Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. Electrophoresis is a rapid separation technique.Factors governing the migration of ions are Charge of ions, Size of the ions, Viscosity of the medium, Voltage applied, pH of buffer and ionic strength. Factors affecting electrophoresis are Molecular characteristics (size, shape), Buffer Properties, Electric Field Characteristics, Temperature.

Reference Id: PHARMATUTOR-ART-1617

I. INTRODUCTION:
FACTORS GOVERNING THE MIGRATION OF IONS:

1.      Charge of ions
2.      Size of the ions
3.      Viscosity of the medium
4.      Voltage applied
5.      pH of buffer and ionic strength

1. Charge of ions:
The electrophoretic mobility is directly proportional to the charge of the molecule, which means that mobility of a molecule is higher when its charge is higher.

2 Size of the ions:
The electrophoretic mobility is inversely proportional to its size, i.e.the mobility is more, when the size is less and vice versa. The mobility is also depends on the shape of the molecule.

3. Viscosity of the medium:
The electrophoretic mobility is directly proportional to the viscosity of the medium.

4. Voltage applied:
Higher the voltage applied faster the separation and sharp bands are obtained. However steps have to be taken to prevent evaporation of the solvent / buffer due to the heat generated by high voltage.

5. pH of Buffer and ionic Strength:
The pH of the buffer solution is to be used is depends on the nature of the components to be separated, in which they can dissociate into ions. Migration of compounds is inversely proportional to the ionic strength. At low ionic strength, migration is faster, but the separated bands appear diffuse. Usually ionic strengths (IS) of 0.05-0.5 is used in more separation.

FACTORS AFFECTING ELECTROPHORESIS:
Electrophoresis is a phenomenon, where charged particles migrate in the presence of an electric field. Biologists use electrophoresis to separate and identify biological molecules such as proteins, nucleic acids and amino acids that exist as either anions or cations. Several factors which affect the electrophoresis are
A)    Molecular characteristics (size, shape)
B)    Buffer Properties
C)    Electric Field Characteristics
D)    Temperature

II. TYPES OF ELECTROPHORESIS:
1.      Paper Electrophoresis
2.      Affinity Electrophoresis
3.      Capillary Electrophoresis
4.      Dielctrophoresis
5.      DNA Electrophoresis
6.      Gel Electrophoresis
7.      Electroblotting
8.      Electrofocussing
9.      Immuno electrophoresis
10.  Isotachophoresis
11.  Protein Electrophoresis
12.  Pulsed Field Gel Electrophoresis

1. Paper Electrophoresis:
This technique is useful for the separation of small charged molecules such as amino acids and small proteins. A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes. The samples are spotted in the centre of the paper, high voltage is applied and the spots migrate according to their charges. After electrophoresis, the separated components can be detected by a variety of staining techniques, depending upon their chemical identity.

This method also useful for determination of protein isoelectric point.It has very useful application in blood cell separation.

2. Affinity Electrophoresis:

This method include
A) Mobility Shift Electrophoresis
B)   Charge Shift Electrophoresis
C) Affinity Capillary Electrophoresis

The methods are based on changes in the ectrophoretic pattern of molecules through bio specific interaction or complex formation. This method has been used for determination of a constants. Affinity electrophoresis may be used as an alternative quantification of the proteins.

3. Capillary Electrophoresis:
It is also known as Capillary Zone Electrophorsis (CZE). This can be use to separate ionic species by their charge and frictional forces and hydrodynamic radius. It can be performed in a capillary format. A typical system consists of two reservoirs and a capillary filled with a buffer solution. A high voltage is applied across the capillary by using a high-voltage power supply. The very small diameter capillaries (typically 5-100 µm) employed in this technique allow for efficient heat dissipation.

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4. Dielectrophoresis (DEP):
It is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to non-uniform electric field. This force doesn’t require the particle to be charged. All particles exhibit dielectrophoretic activity in the presence of electric fields. However the strength of field depends on the medium and particles and on the particle’s shape, size as well as frequency of the electric field.

5. DNA Electrophoresis:
It is an analytical technique used to separate DNA fragments by size. DNA molecules which are to be analysed are set upon a viscous medium, the gel, where electric field induses the DNA to migrate towards the anode, due to the net negative charge of the sugar-phosphate back-bone of the DNA chain. The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel.

6. Gel Electrophoresis:
It refers to using a gel as an anticonvective medium and/or sieving medium during electrophoresis. Gel electrophoresis is most commonly used for separation of biological macromolecules such as DNA, RNA or proteins. It refers to the movement of charged particles in an electric field. Gel suppress the thermal convection caused by application of the electric field, and can act as a sieving medium, retarding the passage of molecules, gel also can simply to serve to maintain the finished separation, so that a post electrophoresis stain can be applied.

7. Electro blotting:
It is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids on to a membrane by using PVDF o\r nitrocellulose, after gel electrophorsis. The protein or nucleic acid can then be further analysed using probes such as specific antibiotics, ligands like lectins or stain. This method can be used with all polyacrylamide and agarose gels. An alternative technique for transferring proteins from a gel is capillary blotting.

8. Electro focussing:
Isoelectro focussing (IEF), also known as electro focussing, is a technique for separating different molecules by their electric charge differences. It is a type of zone electrophoresis, uaually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. Living eukaryotic cells perform isoelectric focussing of proteins.

9. Immunoelectrophoresis:
It is also called as Gamma Globulin Electrophoresis or Immunoglobulin Electrophorsis, is a method of determining the blood levels of three major immunoglobulins: IgM, IgG, IgA.it is a powerful analytical technique with high resolving power as it combines separation of antigens by electrophoresis with immuno diffusion against an antiserum. The increased resolution is of benefit in the immunological examination of serum proteins. This method aids in the diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system.

10. Isotachophoresis:
It is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis. It is a powerful separation technique using discontinuous electrical field to create sharp boundaries between the sample constituents.

In this technique the sample is introduced between a fast leading electrolyte and a slow terminating electrolyte. After application of an electric potential a low electrical field is created in the leading electrolyte and a high electrical field in the terminating electrolyte. The pH at sample level is determined by the counter ion in the leading electrolyte that migrate in the opposite direction. In the first stage the sample constituents migrate at different speeds and start to separate from each other. The faster constituents will create a lower electrical field in the leading part of the sample zone and vice versa.

11. Serum Protein Electrophoresis:
In the bottom plot, a sharp spike has replaced a diffuse hump in the gamma region, because normal gamma globulins are decreased and large amounts of a single monoclonal immunoglobulins have taken their place. Because the protein is monoclonal, each molecule has identical electrophoretic qualities that move it to the same place in the electric field, creating a narrow, intense band or spike. Discovery of an M-Component suggests the presence of multiple myeloma, monoclonal gammopathy of undetermined significance, Waidenstrom’s macroglobulinemia, other lymph proliferative disease, or primary systemic amyloidosis.

12. Pulsed Field Gel Electrophoresis (PFGE):
It is a technique used for the separation of large DNA molecules by applying an electric field that periodically changes direction to a gel matrix.

The theory behind why PFGE works pertains to the mobility of larger DNA fragments. While in general small fragments can find their way through the matrix more easily than large DNA fragments, a threshold length exists above 30-50 kb where all large fragments will run at the same rate and appear in a gel as a single large diffuse band.

III.APPLICATIONS:
Electrophoresis is mainly used for the separation of ionisable substances by using buffers of different pH and ionic strength.
*  Separation of amino acids (into acidic or basic or zwitter ionic type)
*  The type of protein and the percentage of each component can be determined using densitometer.
*  Separation of lipoproteins in serum (in case of hyperlipidemia).
*  Separation of enzymes from blood.
*  For the separation of ions containing same electrophoretic mobility, Isotachophoresis is used to separate the ions by gradient pH application.
*  Dielectrophoresis can be used to manipulate, transport, separate and sort different types of particles.
*  Separation of proteins in serum (into albumin, α1, α2, β1, β2 and Gamma globulins).
*  Separation of alkaloids and antibiotics in different samples can be carried out.
*  For DNA separation and analysis.
*  Vaccine analysis

IV.CONCLUSION:
Electrophoresis is laboratory separation technique which is widely used in biochemistry, biotechnology and analytical laboratories. It is an effective separation technique with the exemption of ‘electro non conductible’ and ‘non-ionic substances’. Now a days it is extensively used for separation and analysis of nucleic acids like DNA and RNA.

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