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Electrophoresis, a method that separates macromolecules in a fluid or gel based on their charge, binding affinity, and size under an electric field, was first observed by Ferdinand Frederic Reuss from Moscow State University in 1807. Anaphoresis and cataphoresis refer to the electrophoresis of negatively and positively charged particles respectively. Electrophoresis is widely used in the separation and analysis of biomolecules such as proteins, plasmids, RNA, DNA, and nucleic acids.

Principle of Electrophoresis and Its Varieties:

The premise of electrophoresis is that charged macromolecules move towards either the negative or positive pole in an electric field, based on their charge. For example, nucleic acid, which carries a negative charge, migrates towards the anode.

There are two main types of this technique: slab electrophoresis and capillary electrophoresis.

Let's delve into the types of Electrophoresis:

Capillary electrophoresis
1. Gel electrophoresis
2. Paper electrophoresis
Slab electrophoresis
1. Zone electrophoresis
2. Immunoelectrophoresis
3. Isoelectrofocusing

Steps in Gel Electrophoresis:

Here's a step-by-step guide to DNA electrophoresis.

Step 1: Sample Preparation –

Start by isolating the DNA and preparing the solution by adding a blue dye to easily observe the sample's movement in the gel.

Step 2: Agarose TAE Gel Solution Preparation –

TAE buffer solution creates an electric field during electrophoresis. To prepare the solution, if you need a 1% agarose gel, add 100mL TAE to 1 g of agarose. Increasing the percentage of agarose results in a denser screen. Heat the agarose TAE solution to dissolve the agarose.

Step 3: Casting the Gel –

Pour the agarose TAE solution into a casting tray and let it cool and solidify. A ready-to-use gel slab with wells is now prepared.

Step 4: Setting Up the Electrophoresis Chamber –

Fill a chamber with TAE buffer and place the solid gel in it, ensuring it is near the negative electrode.

Step 5: Loading the Gel –

Load the wells with the DNA sample and a DNA ladder, which acts as a size reference.

Step 6: Electrophoresis Process –

Connect the positive and negative points to the power supply and chamber, and switch it on. The electric field generated causes the negatively charged sample to move towards the positive point and away from the negative electrode .

Step 7: DNA Observation –

Once you see the migration of the blue-colored DNA samples in the gel, switch off the power supply. Remove the gel and place it in the ethidium bromide solution.

Step 8: Image Capture –

Expose the ethidium bromide-stained gel under UV light and take a photograph. DNA bands appear in the lane of the respective well. Also visible is the DNA ladder, allowing for the length of DNA bands to be determined. Below is an image of a completed experiment.

Types of Electrophoresis

Procedure for Immunoelectrophoresis:

Start by preparing an agarose gel on a horizontal glass slide.
Using a sample template, carefully move the wells to the application zone.
Dilute the sample in a 2:3 ratio with the protein solution.
Use a 5 μl pipette to add 5 μl of the sample and control across each slit.
Place the gel in the chamber for electrophoresis, positioning the sample near the cathode side. Carry out the electrophoresis for 20 minutes at 100 volts.
After the electrophoresis, add 20 μl of antiserum in a trough and incubate for 8- 20 hours at room temperature.
Soak the agarose gel for 10 minutes in saline solution, dry it, and wash it twice.
Dry the gel below 70°C and stain it with a protein stain solution for 3 minutes. Decolorize the gel in a destaining solution for 5 minutes.
Once the gel is dry, you can determine the results.

Types of Electrophoresis

For more in-depth knowledge on the principle, procedure, and applications of other electrophoresis methods, consider enrolling in a comprehensive course.

Frequently Asked Questions

What is Electrophoresis?

Electrophoresis is a technique used to separate macromolecules in a fluid or gel based on their charge, binding affinity, and size under an electric field.

What are the types of Electrophoresis?

The technique is divided into two types viz slab electrophoresis and capillary electrophoresis. These include subtypes such as gel electrophoresis, paper electrophoresis, zone electrophoresis, immunoelectrophoresis, and isoelectrofocusing.

How is gel electrophoresis conducted?

The procedure involves preparing a DNA sample, an agarose TAE gel solution, casting the gel, setting up the electrophoresis chamber, loading the gel, processing electrophoresis, observing the DNA, and finally exposing the ethidium bromide stained gel under UV light.

What is the procedure for immunoelectrophoresis?

The process involves preparing agarose gel, moving the wells to the application zone, making sample dilution, adding the sample across each slit, positioning the gel in the chamber for electrophoresis, adding antiserum, soaking the gel, drying and staining it, and finally determining the results.

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