Electrophoresis

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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ELECTROPHORESIS

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

An electric field is applied across a gel or

The buffer solution maintains a consistent pH

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

Commonly used for separating proteins and

Prepared By - Gaurav Sharma

5. TWO DIMENSIONAL GEL

Prepared By - Gaurav Sharma

Samples are mixed with a loading buffer that

2. LOADING THE SAMPLE

The prepared samples are loaded into wells at

3. RUNNING THE GEL

An electric current is applied, causing the

Prepared By - Gaurav Sharma

After electrophoresis, the gel is stained to

Prepared By - Gaurav Sharma

Digesting DNA with restriction enzymes and

DNA Fingerprinting: Analyzing STR (Short Tandem

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

Preparation and running times can be long,

Primarily qualitative, though quantification is

Risk of contamination or degradation,

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

Prepared By - Gaurav Sharma

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