Electrophoresis: Principle, Types & Procedure

Electrophoresis is a method that is used to separate the molecular components of a sample. It is used in the analysis and separation of macromolecules in a gel or a fluid based on their charge, binding affinity, and size under an electric field.

• Electrophoresis was discovered by Ferdinand Frederic Reuss in 1807 at Moscow State University.
• It is used to separate out DNA, RNA, or protein molecules based on their electrical charge and binding affinity.
• Electrophoresis of negatively charged particles (anions) is known as Anaphoresis.
• Cataphoresis is the electrophoresis of positively charged particles (cations).

Key Terms: Electrophoresis, Electric Field, DNA, RNA, Protein Molecules, RNA, Plasmids, Gel Electrophoresis, Macromolecules

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What is Electrophoresis?

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Electrophoresis is an electrokinetic separation process used for the separation of charged particles present in a fluid through the use of an electric field.

• It is the most common process used for the separation of DNA or protein molecules.
• It can be attained through variegated procedures depending on the size and type of the molecules.
• A support medium, buffer solution, and a source for the electric charge are needed to perform electrophoresis.
• Electrophoresis is very useful in laboratory practices for the bifurcation of molecules based on density, size, and purity.
• It is used in protein electrophoresis as well as plasmids, DNA, nucleic acids, and RNA analysis.

Electrophoresis

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Principle of Electrophoresis

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Electrophoresis is a laboratory technique that works on the principle of electromagnetism.

• The macromolecules placed in an electric field move in the direction of the negative or positive pole as per their charge.
• As nucleic acid is a negatively charged particle, it moves toward the anode. 
• If molecules are positively charged then they will move toward the cathode.
• If the molecules are negatively charged then they will move toward the anode.

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Electrophoresis Process

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Electrophoresis is used to fractionate protein molecules, DNA, or RNA based on the density, type, size, and electrical charge by application of electric current and a gel.

• The pores present in the gel act as a sieve allowing the large molecules to move slower than the small molecules.
• The conditions used during the electrophoresis are altered depending on the desired size range to separate the molecules.
• As the molecules contain an electric charge, thus, when they are subjected to an electric field, a force starts acting upon them.
• The force applied by the electric field increases as the charge in the molecules increases.
• Therefore, depending on the mass of the molecule it starts to move through the support medium.

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Types of Electrophoresis

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The technique of electrophoresis is divided into two major types which have further sub-divisions:

• Capillary Electrophoresis
  • Gel Electrophoresis
  • Paper Electrophoresis
• Slab Electrophoresis
  • Immunoelectrophoresis
  • Isoelectrofocusing
  • Zone Electrophoresis

Capillary Electrophoresis 

Capillary Electrophoresis is used in very small capillaries and microfluid channels. In this method, particles migrate through an electrolytic solution under the influence of an electric field. It is further subdivided into two types namely:

• Gel Electrophoresis: It is one of the most used electrophoresis techniques. Agarose gel electrophoresis, Polyacrylamide gel electrophoresis, and Starch gel electrophoresis are three different types of gel-based electrophoresis. 
• Paper Electrophoresis: It is a technique used for separating small charged molecules of proteins or amino acids from a sample. 

Capillary Electrophoresis 

Slab Electrophoresis 

Slab electrophoresis is the second type of electrophoresis which is used for separating protein molecules by analyzing the samples using a 1D format. Slab electrophoresis is divided into three major types as follows:

• Zone Electrophoresis: It is a technique for the separation of proteins and nucleic acids. 
• Immunoelectrophoresis: It is a process of a combination of immuno-diffusion and electrophoresis.
• Isoelectrofocusing: It is used for separating charged molecules like proteins and peptides.

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What is Gel Electrophoresis?

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Gel electrophoresis is a type of electrophoresis technique used for separating charged molecules, such as DNA, based on their size.

• The charged molecules travel when an electric current is passed through a gel.
• When an electric current is passed through the gel, it creates a positive charge on one end and a negative charge on the other.
• These charged molecules can be separated by an electric field.
• DNA molecules migrate from the negative to a positively charged electrode in gel electrophoresis.

Gel Electrophoresis and DNA

Electrophoresis is a method that helps to distinguish between different lengths of DNA fragments.

• On passing current, DNA will migrate to the positively charged electrode as it is negatively charged.
• The smaller strands of DNA pass through the gel faster than longer strands.
• DNA can be seen on the gel once it has been separated using dyes, fluorescent tags, or radioactive labels.
• It appears as bands on the gel.
• A DNA marker with known length fragments is passed through the gel at the same time as the samples.
• The length of the DNA fragments can be measured by comparing the bands of the DNA samples with those of the DNA marker.

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Gel Electrophoresis Procedure

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The process of Gel Electrophoresis is described as follows: 

• Separate the DNA by adding a blue dye and prepare a solution so that it becomes convenient to keep track of the sample’s movement in the gel.
• An electric field can be generated with the help of a TAE solution during the electrophoresis process.
• In order to make the solution add 100ml of TAE to 1g of agarose. 
• On heating the agarose TAE solution, the agarose gets dissolved. 
• Cool the solution of agarose TAE and allow it to solidify.
• A gel slab as well as a well is now prepared to be applied in this experiment.
• Place the solidified gel in the chamber after filling it up with the TAE buffer.
• Place the gel near the negative electrode. 

Gel Electrophoresis 

• Use the DNA sample and DNA ladder in order to cover the wells.
• Attach the positive and negative points to the power supply and chamber and then switch on the power.
• Migration can be seen in the DNA sample as a result of the generated electric field.
• The sample carrying the negative charge will move in the direction of the positive point and deflect away from the negative point. 
• Once the blue-colored DNA sample can be seen migrating in the gel switch off the power supply.
• Keep the gel on the solution prepared from ethidium bromide.
• Take a picture of the exposed ethidium bromide after staining it under Ultraviolet light.
• The DNA bands can be seen to appear in the lane of the respective well along with the DNA ladder.
• Hence, the DNA band length can be determined.

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Immunoelectrophoresis

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Immunoelectrophoresis is defined as the process of precipitation in agar under an electric field. It involves the combination of immuno-diffusion and electrophoresis. The term “immunoelectrophoresis” was coined by Grabar and Williams in the year 1953.

The process of Immunoelectrophoresis is described as follows: 

• Prepare the agarose gel on a glass slide.
• Using the sample template, move the wells to the application zone.
• Dilute the sample by 2:3 by adding protein to it.
• Now, a condition of stability of the diluent solution is possible through accurate chemical reactions.
• Add a 5 μl sample in a 5 μl pipette carefully.

Immunoelectrophoresis

• Place the gel in the electrophoresis chamber and place the sample near the cathode side.
• The process of electrophoresis should be carried out for 20 mins at 100 volts.
• Take out 20 μl of antiserum in a trough and incubate it for 8- 20 hours at room temperature.
• Soak the agarose gel for 10 minutes in saline solution, then dry it and wash it twice.
• Dry the agarose gel at a temperature below 70°C and stain it with protein stain solution for 3 minutes.
• Decolorize the gel in a destaining solution for around 5 minutes.
• Once the gel is dried, the results can be determined. 

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Things to Remember

• Electrophoresis is a laboratory technique used for the separation of molecules based on density, size, and purity.
• It is used in DNA analysis, the studying of DNA fragments, protein detection, and the testing of antibodies.
• The technique of electrophoresis works on the principle of electromagnetism.
• Capillary Electrophoresis and Slab Electrophoresis are the two types of electrophoresis.
• Gel Electrophoresis is the most widely used electrophoresis technique. 
• Electrophoresis devices render two essential functions namely adsorption of macromolecules for separation and molecular sieving.
• The majority of supporting mediums in electrophoresis include starch, agarose, and polyacrylamide.

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Previous Year Questions

1. Gel electrophoresis is used for the… (AIPMT 2008)
2. In gel electrophoresis, DNA molecules can be separated by… (TS EAMCET 2018)
3. DNA strands on a gel stained with ethidium bromide when viewed… (NEET 2021)
4. Which of the statements is correct in the context of observing DNA separated…
5. Agarose extracted from seaweeds finds used in… (NEET 2011)
6. Which one of the following equipment is essentially required… (NEET 2019)
7. Spooling is… (NEET 2020)
8. Which vector can clone only a small fragment of DNA… (AIPMT 2014)
9. DNA ligase helps in the… (COMEDK UGET 2006)
10. Electroporation Procedure involves… (DUET 2009)