ELECTROPHORESIS

Submitted to: Submitted by:

Dr. Krishan Kant Yashika Rani
Associated Professor PhD coursework (2293)
Department of Microbiology Department of Microbiology
A laboratory technique which is used to separate and analyse biological molecules such as
DNA, RNA and Proteins based on their size, charge and shape under the influence of an
electric field.

Basic mechanism

It involves the movement of charged particle in an electric field, with the separation of
molecules based on their size, shape, and charge in a conductive medium such as gel
matrix or capillary tube.
GENERAL REQUIREMENTS

 Conductive medium

while performing electrophoresis we require a conductive medium such as a buffer solution or a gel
matrix through which the electric field can be applied.
 Power source

A power supply is required to apply an electric field to the conductive medium.

 Electrophoresis apparatus

It is used to hold the conductive medium and sample being analyzed.

It may consist of a gel box, a capillary tube or a microfluidic chip, depending on the type of
electrophoresis being performed.
 Sample

A sample of biomolecule of interest like DNA, RNA or Proteins is required.

 Staining reagents

Staining reagents like Ethidium Bromide for DNA or Coomassie Blue for protein is required to
visualize the molecule.
 Molecular weight markers

Molecular weight markers of known size are often used as a reference for determining the size
of separated molecule.
 Safety equipment

Appropriate safety equipment should be worm when handling sample and reagents.

Apart from these basic requirements the specific requirements for

electrophoresis may vary depending on the type of electrophoresis being
performed and the equipment and reagent used.
GENERAL STEPS
 Preparation of gel:

Agarose gel – dissolve the agarose powder in a buffer solution, heat the solution to dissolve the
agarose then cool the solution to appropriate temperature, and pour the gel into a casting tray.
Polyacrylamide gel – mix the acrylamide and crosslinker solution, add a polymerize initiator and
pour the gel into a casting tray.
 Loading of sample:

Mix the sample with loading buffer and load it onto the gel using micropipette or a gel loading tip.
 Running the gel:

Place the gel in the electrophoresis apparatus and fill the apparatus with a appropriate buffer solution.
Connect the power supply and run the gel at the appropriate voltage nd time to allow thw molecules
to separate based on their size and charge.
 Staining the gel:

Remove the gel from electrophoresis apparatus and stain the gel with a staining reagent.
ETHIDIUM BROMIDE – DNA
COOMASSIE BLUE – PROTEIN
 Visualization:

Observe the separated molecules under UV light for DNA or VISIBLE light for PROTEIN.
These will appear as distinct band of different size and colour.
 Analysis:

Analyse the resulting banding pattern to identify and quantify the separated molecules.

Specific steps for electrophoresis may vary depending on the type

of electrophoresis being performed.
TYPES
 Gel electrophoresis :

Most common type of electrophoresis used to separate molecules based on their size and charge.
Gel electrophoresis can be further divided into :
o Agarose gel electrophoresis –

Used to separate DNA molecules based on their size.

o Polyacrylamide gel electrophoresis –

Used to separate proteins and smaller DNA fragment based on their size.
 Capillary electrophoresis :

This type of electrophoresis separates molecules in a narrow capillary

tube using an applied electric field.
It is often used for high DNA sequencing, genotyping, and analysis of
small molecules.

 Isoelectric focussing :
It separate protein based on their Isoelectric points.

 Two dimensional gel electrophoresis :

It combines two separate electrophoresis runs to
separate proteins based on both their charge and
size.
 SDS – PAGE :

Sodium Dodecyl sulphate Polyacrylamide Gel Electrophoresis is used to

separate proteins based on their size. SDS denatures proteins and gives them
a uniform negative charge, allowing for separation based solely on size.

 Native-PAGE :
It is used to separate proteins based on their size and
charge under native conditions, meaning without
denaturation.

 Pulse-field gel electrophoresis :

It is used to separate large DNA molecules such as
chromosomes based on their size and shape.
 Immuno electrophoresis :
It combines electrophoresis and immunodiffusion to
separate and identify proteins based on their antigenic
properties.
APPLICATIONS

 Biochemistry and molecular biology

 Clinical chemistry
 Forensic science
 Food science
 Environmental science
 Pharmaceutical science
 Quality control
THANK
YOU