Introduction

Electrophoresis is a scientific laboratory technique that is used to separate DNA, RNA, or protein molecules based on their size and electrical charge. An electric current is passed through the molecules to move them so that they can be separated via a gel. The pores present in the gel work like a sieve, allowing smaller molecules to pass through more quickly and easily than the larger molecules. According to the way conditions are adjusted during electrophoresis, the molecules can be separated in the desired size range.

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What is electrophoresis and what are its uses?

Electrophoresis is a very broadly used technique that, fundamentally, applies electric current to biological molecules – they’re usually DNA, but they can be protein or RNA, too – and separates these fragments into pieces that are larger or smaller in size. 

The phenomenon of electrophoresis was first observed by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss in 1807 at Moscow University. A constant application of electric field caused the particles of clay dispersed in water to migrate, showing an electrokinetic phenomenon.

Electrophoresis can be defined as an electrokinetic process that separates charged particles in a fluid using an electrical field of charge. Electrophoresis of cations or positively charged ions is sometimes referred to as cataphoresis (or cataphoretic electrophoresis). In contrast, sometimes, the electrophoresis of anions or negatively charged ions is referred to as anaphoresis (or anaphoric electrophoresis).

It’s used in a variety of applications. Though it is most often used in life sciences to separate protein molecules or DNA, it can be achieved through several different techniques and methods depending upon the type and size of the molecules.

The methods differ in some ways, but all we need is a source for the electrical charge, a support medium and a buffer solution. Electrophoresis is also used in laboratories for the separation of molecules based on their size, density and purity.

The method used to separate macromolecules such as DNA, RNA, or protein molecules is known as gel electrophoresis.

It is used in forensics for – 

• Nucleic acid molecule sizing
• DNA fragmentation for southern blotting
• RNA fragmentation for northern blotting
• Protein fragmentation for western blotting
• Separation of PCR products analysis
• Detection and analysis of variations or mutations in the sequence

Its clinical applications involve – 

• Serum protein electrophoresis
• Lipoprotein analysis
• Diagnosis of haemoglobinopathies and hemoglobin A1c
• Determination of serum protein Phenotypes and micro heterogenesis
• Protein genotyping – e.g., ApoE analysis for Alzheimer’s disease (polymorphic protein)
• Monitoring of Small molecules – e.g., drugs and steroids
• Cerebrospinal fluid analysis
• Urine analysis – like determination of GNs

Principle of electrophoresis

The fundamental principle of electrophoresis is the existence of charge separation between the surface of a particle and the fluid immediately surrounding it. An applied electric field acts on the resulting charge density, causing the particle to migrate and the fluid around the particle to flow.

It is the process of separation or purification of protein molecules, DNA, or RNA that differ in charge, size, and conformation.

The charged molecules are placed at one end of the field according to their charge, and an electric field is applied. 

On passing electric current, depending upon the kind of charge the molecules carry, they move towards the opposite electrodes – either cathode (negative electrode) or anode (positive electrode).

The size, shape, and charge of the molecule remains constant during electrophoresis and determines ionic particle mobility.

An ampholyte acts as a positively charged particle or ion migrating towards the cathode in acidic conditions. In contrast, it acts as a negatively charged particle or ion in an alkaline condition and moves towards the anode.

The rate of migration of an ion in a supporting medium under the influence of an electric field depends upon the following factors – 

• The net charge of the molecule
• The size and shape of the molecule
• The strength of the electric field
• The properties of the supporting medium
• The temperature of the procedure

Factors affecting electrophoresis

Inherent factors

• Magnitude of the charge of a molecule
• Charge density of the molecule
• Molecular weight
• Its shape, i.e., whether it’s tertiary or quaternary structured

External environment

• The pH of the solution
• Electric field
• The viscosity of the solution or agarose concentration
• The temperature of operation

Types of electrophoresis

There are mainly two types of electrophoresis:

 Zone electrophoresis and moving boundary electrophoresis. These two are further classified into – 

Zone electrophoresis :

• Paper electrophoresis
• Gel electrophoresis
• Thin layer electrophoresis
• Cellulose acetate electrophoresis

Moving boundary electrophoresis :

• Capillary electrophoresis

• Isotachophoresis
• Isoelectric Focusing
• Immuno-electrophoresis

Classification of electrophoresis

It is classified into the following:

Slab gel electrophoresis – A traditional method that uses a rectangular gel regardless of its thickness.

Disc electrophoresis – Discontinuities are caused in the electrophoretic matrix because of layers of polyacrylamide or starch gel that differs in composition & pore size.

Isoelectric focusing electrophoresis – IEF separates atmospheric compounds (like proteins) with increased resolution in a medium possessing a stable pH gradient.

Isotachophoresis – It completely separates smaller ionic substances into adjacent zones that contact one another without overlapping & all of them have the same rate of migration.

Pulse-Field electrophoresis – Power is alternatively applied to different pairs of electrodes or electric arrays such that the electrophoretic field is cycled between two directions.

2D electrophoresis is a charge-dependent IEP in the first dimension, whereas it is molecular weight dependent in the second dimension.

Conclusion

Electrophoresis can be considered as an electrokinetic phenomenon involving an electric field, supporting medium, and a buffer solution. It has many forensic and clinical applications in our daily life. From using traditional methods of only rectangular gel usage to using enhanced resolution techniques, electrophoresis has come a long way since its discovery in 1807 in Russia. Overall, electrophoresis is a separation technique that can single out biomolecules or charged particles of interest based on their mobility in a given electric field. Gel electrophoresis allows scientists to visualise the sizes of DNA segments and aids in the sequencing of lengths of DNA.