ELECTROPHORESIS

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 What is Electrophoresis ?
Electrophoresis is a laboratory technique used
to separate and analyze macromolecules
(such as DNA, RNA, and proteins) based on
their size, charge, and other physical
properties. The process involves applying an
electric field to a gel matrix or other medium,
causing the molecules to migrate at different
rates. This technique is fundamental in various
fields, including molecular biology,
biochemistry, genetics, and forensic science.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Key Components of
Electrophoresis
1. ELECTRIC FIELD

An electric field is applied across a gel or

another medium, causing charged molecules
to migrate toward the electrode of opposite
charge. Positively charged molecules move
toward the cathode (negative electrode), and
negatively charged molecules move toward
the anode (positive electrode).

2. BUFFER

The buffer solution maintains a consistent pH

and conducts the electric current through the
gel.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 3. GEL MATRIX
The gel, often made of agarose or
polyacrylamide, acts as a sieve, allowing
smaller molecules to move faster and larger
molecules to move slower. This differential
movement enables separation based on size.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Types of Electrophoresis
1. AGROSE GEL ELECTROPHORESIS
Primarily for separating nucleic acids (DNA and
RNA).
Gel made from agarose, a polysaccharide
extracted from seaweed.
Suitable for separating large fragments of
nucleic acids (hundreds to thousands of base
pairs).
Stained with intercalating agents like ethidium
bromide or SYBR Green, which fluoresce under
UV light.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 2. POLYACRYLAMIDE GEL
ELECTROPHORESIS

Commonly used for separating proteins and

small nucleic acids. Gel made from acrylamide
and bisacrylamide, which form a cross-linked
matrix. High resolution, suitable for small
fragments (as small as single base
differences).
Variants: SDS-PAGE: Denaturing conditions
using Sodium Dodecyl Sulfate (SDS) to
separate proteins based on size.
Native PAGE: Non-denaturing conditions to
separate proteins based on size, shape, and
charge.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 3. CAPILLARY ELECTROPHORESIS
High-resolution separation of nucleic acids, proteins,
and small molecules. Uses a thin capillary tube filled
with a gel or buffer solution. Fast, highly automated,
and requires small sample volumes.

4. ISOELECTRIC FOCUSING
Separating proteins based on their isoelectric point
(pI). Uses a pH gradient within a gel. Proteins migrate
to the pH corresponding to their pI, where they have
no net charge and stop moving.

5. TWO DIMENSIONAL GEL

ELECTROPHORESIS
Separating complex protein mixtures. Combines IEF
(separation by pI) in the first dimension with SDS-
PAGE (separation by size) in the second dimension.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Procedure
1. SAMPLE PREPARATION

Samples are mixed with a loading buffer that

often contains tracking dyes, density-
increasing agents like glycerol, and in some
cases, denaturing agents (e.g., SDS for
proteins).

2. LOADING THE SAMPLE

The prepared samples are loaded into wells at

one end of the gel.

3. RUNNING THE GEL

An electric current is applied, causing the

samples to migrate through the gel matrix. The
migration is monitored using tracking dyes.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 4. STAINING & VISUALISATION

After electrophoresis, the gel is stained to

visualize the separated molecules. Common
stains include ethidium bromide for nucleic
acids and Coomassie Brilliant Blue or silver
stain for proteins.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Applications of
Electrophoresis
1. DNA & RNA ANALYSIS

Digesting DNA with restriction enzymes and

separating fragments to create a DNA
fingerprint.
Checking the size and purity of PCR-amplified
DNA.
Assessing RNA quality and integrity for
subsequent experiments.

2. FORENSIC SCIENCE

DNA Fingerprinting: Analyzing STR (Short Tandem

Repeats) for identity verification.Crime Scene
Analysis: Comparing DNA samples from crime
scenes with suspects.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 3. PROTEIN ANALYSIS
Assessing the purity of protein samples.
Protein Identification: In combination with
techniques like mass spectrometry.
Transferring proteins from a gel to a
membrane for immunodetection.

4. CLINICAL DIAGNOSTICS
Detecting mutations associated with genetic
diseases.
Identifying bacterial or viral DNA/RNA in
patient samples.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Advantages of
Electrophoresis
1. VERSATILITY
Applicable to a wide range of biological
molecules.
2. RESOLUTION
High resolution allows separation of molecules
with very small differences in size or charge.
3. SIMPLE & COST EFFECTIVE
Many electrophoresis techniques are
straightforward and require basic laboratory
equipment.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 Limitations of
Electrophoresis
1. TIME CONSUMING

Preparation and running times can be long,

especially for complex gels.

2. LIMITED QUANTITATIVE
INFORMATION

Primarily qualitative, though quantification is

possible with additional techniques.

3. SAMPLE HANDLING

Risk of contamination or degradation,

especially for sensitive samples.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
The Bottom Line
Electrophoresis is a cornerstone
technique in molecular biology and
biochemistry, providing essential
capabilities for the separation and
analysis of macromolecules. Its
diverse applications, from genetic
analysis and protein
characterization to forensic
investigations and clinical
diagnostics, underscore its
significance in scientific research
and practical applications.

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer
 "Let's Connect!"

Prepared By - Gaurav Sharma

Food Technologist/Academic Writer