Types of Electrophoresis: Capillary and Slab Electrophoresis

Electrophoresis is a method of separating the molecular components of a sample. It is used for DNA, RNA, protein molecules or any other molecules. The method allows to separate out DNA, RNA or protein molecules based on their electrical charge and binding affinity. Electrophoresis was first observed by Ferdinand Frederic Reuss in 1807 at Moscow State University. Electrophoresis of negatively charged particles (anions) is known as Anaphoresis and positively charged particles (cations) are known as Cataphoresis. Electrophoresis can be used in the analysis and separation of the macromolecules present in fluid or gel based on the charge under an electric field.

Key Terms: Electrophoresis, DNA, RNA, Gel Electrophoresis, Paper Electrophoresis, Zone Electrophoresis, Immunoelectrophoresis, Slab Electrophoresis, Electromagnetism

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Principle of Electrophoresis

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Under the influence of electric potential, the macromolecules placed in an electric field moves in the direction of the negative or positive pole. As nucleic acid is a negatively charged particle, so it moves toward the anode. 

The entire electrophoresis process has two varieties: slab electrophoresis and capillary electrophoresis. If molecules are positively charged then they will move towards the cathode and if the molecule is negatively charged then it will move towards the anode.

Electrophoresis works on the fundamental equation of the science of electromagnetism.

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Types of Electrophoresis

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Electrophoresis is divided into two types mainly: 

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

Read More: Cathode Ray Discharge Tubes

Capillary Electrophoresis 

It is an electrokinetic separation method which is performed in very small capillaries and micro fluid channels. Basically, CE stands for capillary zone electrophoresis (CZE) and the other techniques of electrophoresis are micellar electrokinetic chromatography, gel electrophoresis, capillary isoelectric focusing, etc. 

In the CE method, particles migrate through an electrolytic solution under the influence of an electric field. It is of 2 types: gel electrophoresis and paper electrophoresis.

Gel Electrophoresis

It is one of the most used electrophoresis procedures in a variety of experimental environments. There are three different types of gel-based electrophoresis. These are agarose gel electrophoresis, polyacrylamide gel electrophoresis, and starch gel electrophoresis. Now, we will study the steps involved during DNA electrophoresis:

1. Preparation of sample: The DNA is extracted from the sample and the solution is prepared by adding blue dye so that we can easily observe the movement of the sample taking place in the gel.
2. Preparation of an agarose TAE gel solution: The use of a TAE buffer solution to generate the electric field is one of the most efficient ways in the process of electrophoresis. To prepare a solution of 1% agarose gel, we need to add 100 ml of TAE to 1 g of agarose. Then heat the solution for proper dissolution of agarose in agarose TAE solution.
3. Gel casting: Pour a fresh agarose TAE solution onto the casting tray, allow it to solidify and then scrape off the flats that remain. Now, this slab along with the wells is ready to use in the experiment.
4. Setup of electrophoresis chamber: Fill the TAE buffer in the chamber and place the solid gel in the chamber in such a way that it is located near the negative chamber.
5. Gel loading: Load the wells with a DNA sample.
6. Main process of electrophoresis takes place: Connect the positive and negative terminals carefully and switch on the power. Due to the generation of the electric field, the DNA sample will start to migrate. The negatively charged sample will move towards the positive terminal and the positively charged sample will move towards the negative terminal.
7. Observe the DNA carefully: Switch off the power supply immediately when you will observe the movement of a blue-coloured DNA sample in the gel. Now carefully remove the gel and place it in the solution of ethidium bromide.
8. Expose the ethidium bromide-stained gel under UV light and take a picture. DNA bands appear in the lane of the respective well.
9. Hence from the experiment, we can determine the length of DNA bands.

Gel Electrophoresis

Read More: Principle of UV Visible Spectroscopy

Paper Electrophoresis

Paper electrophoresis is a simple technique for separating small charged molecules of proteins or amino acids from a sample. The process starts with applying the sample to the strips of paper and then moisturizing it using a buffer solution. Each end of the paper is dipped in different tanks of buffer solution. 

Now the electric field is applied and the charged particles of the sample will start moving towards the opposite polarity of the electrodes. After the completion of the process, the paper is dried and then observed properly using a sound quality detection system.

Slab Electrophoresis

Slab electrophoresis is a method of separating protein molecules by analyzing the samples using a 1D format. There is no requirement for instrumentation in SE as it was used in CE. There are three types of slab electrophoresis: Zone electrophoresis, Immunoelectrophoresis, and Isoelectrofocusing.

Immunoelectrophoresis

1. Firstly, agarose gel is prepared on a glass slide.
2. Use the sample template and carefully move the wells to the application zone.
3. Diluting the sample by 2:3 by adding protein to it. Maintaining a condition of stability of the diluent solution through accurate chemical reactions is now possible.
4. Add 5 μl sample in 5 μl pipette carefully.
5. Now place the gel in the electrophoresis chamber and place the sample near the cathode side.
6. Carry out the electrophoresis at 100 volts for 20 minutes.
7. Take 20 μl of antiserum in a trough and incubate for 8- 20 hours at room temperature on competing for the electrophoresis.
8. Soak the agarose gel for 10 minutes in saline solution, dry it and wash it twice.
9. Dry the gel below 70°C and stain it with protein stain solution for 3 minutes.
10. Decolourize the gel in a destaining solution for 5 minutes.
11. Determine the results once the gel is dried.

Immunoelectrophoresis

Read More: SDS PAGE

Isoelectrofocusing

It is a technique of separating charged molecules like proteins and peptides. The separation depends upon the pH of the uncharged molecule and its isoelectric point. The molecules under their pH gradient tend to move towards their pI in an electric field.

Zone Electrophoresis

Zone electrophoresis is a technique for the separation of proteins and nucleic acids. The separation is carried out by applying the electric current in the buffer system and the transportation of different species will start. Because of the different mobilities, these species will subdivide into small peaks or regular dispersal patterns.

Read More: Biomolecules

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Things to Remember

• Electrophoresis is a technique that allows the separation of DNA from different types of nucleic acids.
• Electrophoresis is also defined as a technique that allows the separation of two or more sample solutions to determine the concentration of an analyte in a sample.
• Under the influence of electric potential, the macromolecules placed in an electric field move in the direction of the negative or positive pole.
• Electrophoresis works on the fundamental equation of the science of electromagnetism.
• Capillary electrophoresis (CE) is an electrokinetic separation method that is performed in very small capillaries and microfluid channels.
• CE is of two types: gel electrophoresis and paper electrophoresis.
• Slab electrophoresis (SE) is a method of separating protein molecules by analyzing the samples using a 1D format.
• SE is of three types: zone electrophoresis, isoelectric focusing, and Immunoelectrophoresis.

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Important PYQs-Based Electrophoresis

1. Which One Of The Following Graphs Represents The V
2. Two Point Charges Q And 3q Are Placed Certain Dist
3. Two Point Charges Of 1 C And 1 C Are Separated By
4. Two Charges Q And 3q Are Fixed On X Axis Separated
5. Three Charge Q Q And 4q Are Placed In A Straight L
6. The Unit Of Permittivity Is Same As That Of
7. The Nucleus Of Helium Atom Contains Two Protons Th
8. The Force Per Unit Charge Is Known As
9. The Electric Field Strength At A Distance R From A
10. The Electric Field Intensity At A Point P Due To P
11. The Electric Field Components In The Given Figure
12. The Electric Field At A Point Is
13. Two Parallel Large Thin Metal Sheets Have Equal Su
14. Two Point Charges Q 1 2 C And Q 2 1 C Are Placed A
15.  The Positive Charged Ball Is Suspended By Thread O… [RPET 2002]
16. The Magnitude Of Electric Field Intensity E Such T
17. The Displacement Of A Charge Q In The Electric Fie
18. An Electric Line Of Force In The Xy Plane Is Given
19. Two Point Charges 10 7 C And 10 7 C Are Placed At