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Chapter 4:Electrophoresis

Long Answer Questions

1. Write in detail about the gels used in electrophoresis

Gels Used in Electrophoresis: An In-Depth Exploration

Electrophoresis is a laboratory technique that separates molecules, such as proteins,

nucleic acids, and other biomolecules, based on their size, charge, and other properties,
by applying an electric field to a gel matrix. The gel acts as a medium that supports the
migration of molecules during the electrophoretic process, providing the necessary
resistance to allow separation. The gel matrix also facilitates the visualization of the
separated biomolecules, making it possible to analyze and interpret experimental results.
The choice of gel type and composition Is critical for the success of electrophoresis
experiments. In this article, we will delve into the different types of gels used in
electrophoresis, their properties, and their applications.

Types of Gels in Electrophoresis

The most commonly used gels in electrophoresis are agarose and polyacrylamide, though
other materials, such as starch and cellulose, have also been used in specific applications.

1. Agarose Gel

Agarose is a polysaccharide that is derived from seaweed and is widely used in nucleic
acid electrophoresis, particularly in the separation of DNA and RNA molecules. Agarose
gels are simple to prepare and offer several advantages, including excellent resolution and
reproducibility. The gel consists of a network of long agarose molecules that form a three-
dimensional mesh. This network acts as a sieve, with the size of the pores being
determined by the concentration of agarose used in the gel. Higher agarose concentrations
lead to smaller pores, which are ideal for separating smaller molecules, while lower
concentrations are better suited for larger molecules.

Properties of Agarose Gel:

High porosity: The porosity of agarose gels allows the separation of a wide range of
biomolecules based on size.

Biocompatibility: Agarose gels are relatively non-toxic, making them suitable for use in
biological experiments.
Ease of preparation: Agarose gels are prepared by dissolving agarose powder in a buffer
solution, heating it to a boil, and pouring it into a gel tray to solidify.

Applications of Agarose Gel:

Agarose gel electrophoresis is widely used in molecular biology for the analysis of DNA and
RNA. It is commonly employed for:

DNA fragment analysis: Agarose gels are used to separate DNA fragments of varying sizes
after PCR (polymerase chain reaction) amplification, restriction enzyme digestion, or DNA
sequencing.

RNA separation: Agarose gel electrophoresis is useful for separating RNA molecules based
on their size, often in the context of gene expression analysis or RNA isolation.

Southern and Northern blotting: Agarose gel electrophoresis is used to separate DNA and
RNA for subsequent transfer to membranes in Southern and Northern blotting,
respectively.

2. Polyacrylamide Gel

Polyacrylamide is a synthetic polymer that is commonly used for protein electrophoresis

and for separating smaller nucleic acid fragments, such as oligonucleotides. The gel is
formed by polymerizing acrylamide monomers in the presence of a crosslinker (usually bis-
acrylamide), which creates a three-dimensional network of polymers. The pore size of
polyacrylamide gels can be controlled by adjusting the concentration of acrylamide and
bis-acrylamide, allowing for fine-tuned separation of proteins or nucleic acids.

Properties of Polyacrylamide Gel:

Higher resolution: Polyacrylamide gels provide superior resolution compared to agarose

gels, making them suitable for the analysis of smaller molecules, such as proteins.

Adjustable pore size: The pore size of polyacrylamide gels can be adjusted by varying the
concentration of acrylamide and bis-acrylamide.

Chemical stability: Polyacrylamide gels are chemically stable and provide consistent
results over time.

Applications of Polyacrylamide Gel:

Polyacrylamide gel electrophoresis (PAGE) is a versatile technique used for separating

proteins and small nucleic acids. Common applications include:
SDS-PAGE: Sodium dodecyl sulfate (SDS)-PAGE is a method used to separate proteins
based on their molecular weight. The SDS detergent denatures proteins and imparts a
negative charge, causing them to migrate through the polyacrylamide gel according to size.

Native PAGE: In native PAGE, proteins are separated based on their size and charge without
denaturation, allowing for the analysis of protein-protein interactions and functional
studies.

Agarose vs. Polyacrylamide in Nucleic Acid Electrophoresis: While agarose gels are
typically used for large DNA molecules, polyacrylamide gels are more suitable for
separating small DNA fragments, such as oligonucleotides, or for resolving high-resolution
RNA profiles.

3. Starch Gel

Starch gels are occasionally used for electrophoresis, particularly in the analysis of
enzymes and certain proteins. Starch is a natural polymer composed of glucose units, and
starch gels are formed by dissolving starch powder in hot water and cooling it to form a gel
matrix. Starch gels have larger pores than agarose or polyacrylamide gels, making them
suitable for the separation of larger proteins.

Applications of Starch Gel:

Enzyme analysis: Starch gel electrophoresis was once commonly used to study enzymes,
especially for the detection of genetic variants.

Protein analysis: Although less commonly used today, starch gel electrophoresis has been
employed to analyze the molecular weights of certain proteins and their variants.

4. Cellulose Acetate Gel

Cellulose acetate is another material used in electrophoresis, primarily for protein

analysis. The cellulose acetate gel is made from thin sheets of cellulose acetate, which
acts as a medium for protein separation. Cellulose acetate is used in situations where
rapid protein analysis is required, and its advantages include easy handling and the ability
to analyze multiple samples simultaneously.

Applications of Cellulose Acetate Gel:

Protein diagnostics: Cellulose acetate gel electrophoresis has been employed for
diagnostic purposes, particularly in identifying hemoglobin variants or studying enzyme
polymorphisms.
Electrophoretic protein separation: It is used for separating serum proteins, particularly in
clinical laboratories.

Factors Affecting Gel Performance

The performance of an electrophoresis gel is influenced by several factors:

Gel concentration: The concentration of the gel affects its pore size. Higher concentrations
provide smaller pores, which are better for separating smaller molecules, while lower
concentrations are suitable for larger molecules.

Buffer system: The choice of buffer affects the pH and ionic strength, which in turn affects
the mobility of charged molecules during electrophoresis.

Voltage: The voltage applied during electrophoresis determines the speed of migration.
Higher voltages lead to faster migration, but they can also generate heat, which may affect
the integrity of the gel or the biomolecules being analyzed.

2. Discuss the theory of electrophoresis and factors affecting electrophoretic

mobility,
Theory of Electrophoresis and Factors Affecting Electrophoretic Mobility
Electrophoresis is a widely used analytical technique that separates charged
particles, such as proteins, nucleic acids, and other biomolecules, based on their
size and charge under an electric field. The term “electrophoresis” is derived from
two Greek words: “electron,” meaning “amber” (which refers to electricity), and
“phoresis,” meaning “carrying” or “movement.” This technique plays a crucial role in
molecular biology, biochemistry, and biotechnology by providing insights into the
composition, purity, and size of biomolecules.
Basic Principle of Electrophoresis
The basic principle of electrophoresis relies on the movement of charged particles
in an electric field. When a sample is placed in a gel matrix, typically made of
agarose or polyacrylamide, and an electric field is applied, charged molecules will
migrate towards the electrode of opposite charge. The velocity of migration depends
on several factors, including the particle’s size, charge, shape, and the nature of the
medium in which it is placed.
1. Charge: Charged particles move towards the electrode with an opposite charge.
For example, positively charged particles (cations) will migrate towards the
cathode (negative electrode), while negatively charged particles (anions) will
move towards the anode (positive electrode).
2. Electric Field: The electric field is generated by applying a voltage across the two
electrodes. The force exerted on the charged particles is proportional to the
 magnitude of the electric field. The electric field provides the driving force for the
particles to move through the medium.
3. Medium: The medium, typically a gel, provides both resistance and separation of
particles. It creates a physical barrier that affects the speed at which the
particles migrate. The choice of medium is critical and depends on the type of
particles being analyzed.
Electrophoretic Mobility
Electrophoretic mobility is defined as the velocity of a particle in an electric field
divided by the field strength. Mathematically, it is represented as:
Where is the electrophoretic mobility, is the velocity of the particle, and is the
electric field strength. Electrophoretic mobility is typically measured in units of .
Several factors influence the electrophoretic mobility of charged particles,
including:
1. Charge-to-Mass Ratio (Z): The charge-to-mass ratio is a significant determinant
of electrophoretic mobility. Particles with a higher charge-to-mass ratio will
experience a stronger force in the electric field, resulting in higher mobility. This
is why smaller and more highly charged particles tend to move faster than larger,
less charged particles.
2. Shape of the Particle: The shape of a particle can significantly influence its
mobility. Irregularly shaped particles often experience more resistance as they
move through the gel, which decreases their mobility. In contrast, spherical
particles with a uniform shape move more efficiently.
3. Size of the Particle: Larger particles experience greater friction as they move
through the gel matrix, which reduces their mobility. Smaller particles face less
resistance and thus migrate faster. This size-based separation is one of the
primary principles in gel electrophoresis, where smaller molecules travel further
in a given time than larger molecules.
4. Viscosity of the Medium: The viscosity of the gel medium also plays a role in
determining the mobility of particles. Higher viscosity results in more resistance
to particle movement, thereby decreasing the electrophoretic mobility. This is
particularly relevant in the context of the gel concentration and composition
used, as it determines the gel’s porosity.
5. Electric Field Strength €: The strength of the electric field influences how quickly
particles migrate. However, it is important to note that increasing the electric
field strength beyond a certain point can lead to the overheating of the system,
which can affect the stability of the gel and the separation process. Additionally,
very high field strengths may cause distortions in the shape of the particle,
further affecting the migration rate.
6. Temperature: Temperature affects the mobility of particles because it influences
the viscosity of the medium and the kinetic energy of the particles. A higher
temperature can increase the speed at which molecules move, but excessive
heat can cause the gel to melt or lead to unwanted effects like band broadening.
Factors Affecting Electrophoretic Mobility
1. Ion Concentration of the Buffer Solution: The concentration of ions in the buffer
solution affects the conductance of the electric field and can influence the ion
cloud around the charged particle. Higher ion concentration may reduce the
electrical resistance, thus facilitating faster migration of particles.
2. pH of the Buffer Solution: The pH of the buffer solution can impact the charge of
the molecules being analyzed. Many biomolecules, such as proteins, have
functional groups that can gain or lose protons depending on the pH. This leads
to a change in the overall charge of the molecule, which in turn affects its
migration. For example, proteins are negatively charged at high pH (basic
conditions) and positively charged at low pH (acidic conditions).
3. Nature of the Gel Matrix: The type of gel matrix used for electrophoresis is
another critical factor that influences mobility. Agarose and polyacrylamide gels
are most commonly used, and their concentration dictates the pore size of the
matrix. A higher concentration of gel creates a finer matrix, providing greater
separation power but also increasing resistance to particle movement, which
can reduce mobility.
4. Electroosmotic Flow: In capillary electrophoresis, electroosmotic flow refers to
the bulk movement of the buffer solution in response to the applied electric
field. This flow can either aid or hinder the migration of the particles depending
on the direction and magnitude of the flow. In some cases, it can lead to
distortion of the bands or alter the resolution of the separation.
Applications of Electrophoresis
Electrophoresis has a wide range of applications in various fields, including:
Protein and DNA analysis: Electrophoresis is used to separate and analyze proteins
and nucleic acids, providing crucial information about their size, charge, and
composition.
Forensic science: It is used in DNA fingerprinting to identify individuals based on
unique patterns of DNA.
Clinical diagnostics: Electrophoresis is employed to analyze blood serum proteins,
enabling the detection of diseases such as multiple myeloma or sickle cell anemia.
Conclusion
Electrophoresis is a powerful tool for the separation and analysis of charged
particles. The theory behind electrophoresis is rooted in the movement of charged
 molecules under an electric field, with their mobility determined by factors such as
size, charge, shape, and the characteristics of the medium. Understanding the
factors that affect electrophoretic mobility, such as charge-to-mass ratio, gel
composition, and pH, is crucial for optimizing electrophoretic separation
techniques. By manipulating these variables, researchers and clinicians can
achieve precise molecular analysis, contributing to advancements in fields like
molecular biology, biotechnology, and medical diagnostics.

3.Explain in detail the role of SDS in SDS-PAGE with its application

The Role of SDS in SDS-PAGE and Its Applications

Sodium dodecyl sulfate (SDS) is a key component in the technique of SDS-PAGE (Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which is widely used for the
separation and analysis of proteins based on their molecular weight. SDS-PAGE is one of
the most important and versatile methods in molecular biology, biochemistry, and
analytical chemistry. It allows scientists to analyze the protein composition of a sample,
determine protein sizes, and study protein purity. This article discusses the role of SDS in
SDS-PAGE in detail, highlighting its function, mechanism, and applications.

1. What is SDS?

Sodium dodecyl sulfate (SDS) is an anionic detergent with a hydrophobic tail and a
negatively charged sulfate group at the head. It is a surfactant, meaning it can interact with
both lipophilic and hydrophilic environments, which makes it effective in disrupting the
non-covalent interactions between proteins and other macromolecules.

2. The Role of SDS in SDS-PAGE

SDS is the critical reagent in SDS-PAGE for ensuring the separation of proteins based solely
on their size. The mechanism through which SDS operates involves several steps:

a. Denaturation of Proteins: The first key role of SDS is to denature the proteins
in a sample. Native proteins have complex three-dimensional structures,
held together by various non-covalent interactions such as hydrogen bonds,
hydrophobic interactions, and ionic bonds. These interactions can cause the
proteins to fold into specific shapes that are essential for their function.
However, to separate proteins by size in SDS-PAGE, these structures must be
unfolded. SDS binds to the hydrophobic regions of the protein, disrupting
these non-covalent bonds and causing the protein to unfold into a linear
structure. This denaturation ensures that proteins lose their secondary,
tertiary, and quaternary structures.
 b. Binding to Proteins: SDS molecules bind to proteins in a roughly proportional
manner to the protein’s length, with approximately 1 SDS molecule binding
for every 2 amino acids in the polypeptide chain. The result is that the protein
is coated with a negative charge, which makes the overall charge of the
protein independent of its intrinsic properties. This ensures that all proteins
will have a net negative charge, regardless of their amino acid composition,
as long as they are denatured.
c. Imparting Uniform Negative Charge: The binding of SDS molecules to
proteins is crucial because it imparts a uniform negative charge to the
proteins. This uniform negative charge is essential for the separation of
proteins during electrophoresis. Without this step, proteins would have
different charges based on their amino acid sequence, which could lead to
their migration in unpredictable directions during electrophoresis. By adding
SDS, the charge-to-mass ratio of the proteins becomes approximately the
same, ensuring that their movement through the polyacrylamide gel will be
governed primarily by their size, not their charge.

D. Maintaining Protein Solubility: SDS also helps to keep proteins in a soluble state by
preventing aggregation. Proteins can aggregate if they refold incorrectly or interact with
other proteins due to hydrophobic regions becoming exposed. SDS prevents this by
maintaining the proteins in a denatured, linear form and keeping them suspended in
solution during the electrophoresis process.

3. Electrophoresis and Separation Based on Size

Once SDS binds to the proteins and unfolds them into a linear form, the proteins are
loaded into a polyacrylamide gel, which acts as a molecular sieve. When an electric
current is applied, the negatively charged SDS-coated proteins move toward the positive
electrode. Smaller proteins can move through the gel matrix more easily and quickly than
larger ones, leading to their separation based on size.

The polyacrylamide gel provides a medium where proteins are resolved according to their
molecular weight. The separation occurs because the gel’s pore size restricts the
movement of larger molecules, causing them to migrate more slowly than smaller ones. As
a result, after electrophoresis, proteins of different sizes will be distributed in distinct
bands, with smaller proteins at the bottom of the gel and larger ones closer to the top.

4. Applications of SDS-PAGE

SDS-PAGE has numerous applications in molecular biology, biochemistry, and proteomics.

Some of the most important applications are:
 a. Protein Size Determination: One of the primary applications of SDS-PAGE is
determining the molecular weight of proteins. By comparing the migration of
proteins in the sample to a set of molecular weight markers (standard
proteins of known size), researchers can estimate the size of unknown
proteins. This is a fundamental technique for protein identification and
characterizing proteins.
b. Protein Purity Analysis: SDS-PAGE is widely used to assess the purity of
protein samples. Proteins that are highly pure will appear as a single band on
the gel, while impurities (such as other proteins) will result in additional
bands. This is crucial for ensuring the quality of proteins used in research,
industrial applications, or therapeutic treatments.

c. Protein Expression Analysis: SDS-PAGE is used to analyze the expression

levels of recombinant proteins in expression systems like E. coli, yeast, or
mammalian cells. By comparing the expression of the target protein to
control samples, researchers can quantify protein production and evaluate
the success of genetic engineering efforts.

D. Identification of Post-Translational Modifications: SDS-PAGE can be combined with

other techniques such as Western blotting to identify post-translational modifications
(PTMs), such as phosphorylation, glycosylation, or ubiquitination. These modifications
often affect the mobility of proteins during electrophoresis, enabling researchers to
investigate how modifications affect protein function.

d. Detection of Protein-Protein Interactions: SDS-PAGE can also be used to

study protein-protein interactions. For example, by cross-linking interacting
proteins, researchers can identify protein complexes by observing the
migration of multi-protein assemblies in the gel.
e. Analysis of Protein Degradation: SDS-PAGE is useful for studying protein
degradation. Degradation products, which are smaller fragments of the
original protein, will migrate differently than the intact protein, allowing
researchers to analyze the degradation process.
5. Conclusion

SDS plays a crucial role in SDS-PAGE by denaturing proteins, providing them with a uniform
negative charge, and allowing for their separation based on size during electrophoresis. The
use of SDS enables the precise analysis of proteins in terms of size, purity, expression
levels, and modifications, making SDS-PAGE an indispensable tool in modern molecular
biology and biochemistry. Its wide range of applications has made it a standard technique
in protein research, drug development, and various other fields where protein analysis is
required.

4.Describe the process of slab gel electrophoresis

Slab Gel Electrophoresis: An Overview

Slab gel electrophoresis is a widely used laboratory technique for the separation of
macromolecules, such as proteins, nucleic acids (DNA and RNA), and other
biomolecules, based on their size, charge, and conformation. This method relies on
the movement of charged particles in an electric field through a gel matrix, where
the separation is facilitated by the different migration rates of the molecules. Slab
gel electrophoresis is commonly applied in molecular biology, biochemistry, and
clinical diagnostics, among other fields.

Principle of Slab Gel Electrophoresis

The basic principle of electrophoresis is that charged molecules will move in an

electric field. The direction of movement depends on the charge of the molecule:
negatively charged molecules migrate toward the anode (positive electrode), while
positively charged molecules move toward the cathode (negative electrode). The
rate at which a molecule moves through the gel is influenced by several factors,
including its size, shape, and the ionic strength of the buffer solution.

In slab gel electrophoresis, a gel matrix, typically made of agarose (for nucleic acids)
or polyacrylamide (for proteins), is used to separate the molecules. The gel acts as a
molecular sieve, providing a medium that allows smaller molecules to move more
quickly, while larger molecules experience more resistance and migrate more
slowly. This size-dependent separation allows for the characterization and
quantification of biomolecules.

Types of Slab Gel Electrophoresis

Slab gel electrophoresis can be used to separate different types of biomolecules.

The specific type of gel and buffer used depends on the nature of the sample being
analyzed:

Agarose Gel Electrophoresis: Agarose gel is most commonly used for DNA and RNA
separation. Agarose is a polysaccharide extracted from seaweed, and it forms a gel
when dissolved in a buffer and cooled. The agarose gel matrix provides a network of
pores that allows for the separation of nucleic acids based on their size. The DNA or
RNA samples are usually stained with a dye, such as ethidium bromide or SYBR
Green, to make them visible under ultraviolet (UV) light.

Polyacrylamide Gel Electrophoresis (PAGE): Polyacrylamide gel electrophoresis is

typically used for protein separation. Polyacrylamide gels have a finer pore structure
compared to agarose gels, making them suitable for resolving smaller proteins. This
technique is often combined with denaturing agents like sodium dodecyl sulfate
(SDS), which ensures that proteins are separated based on their molecular weight,
rather than their charge or shape.

Components of Slab Gel Electrophoresis

1. Gel Matrix: The gel serves as a medium through which the biomolecules migrate.
The pore size of the gel can be adjusted by varying the concentration of agarose
or polyacrylamide. The gel is usually prepared in a horizontal or vertical format,
depending on the type of electrophoresis being performed.
2. Electrophoresis Buffer: The buffer provides the necessary ions to conduct the
electric current through the gel. Common buffers for nucleic acid
electrophoresis include TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA)
buffers. For protein electrophoresis, a variety of buffers can be used, depending
on the specific requirements of the experiment.
3. Electrode Assembly: Two electrodes (anode and cathode) are placed at opposite
ends of the gel apparatus. The electrodes are connected to a power supply that
generates the electric field necessary for the migration of charged molecules.
4. Sample Loading Wells: The gel has small wells where the sample is loaded.
Typically, a sample buffer is mixed with the biomolecule sample before loading it
into the wells to ensure it has the appropriate consistency for loading and
migration.
5. Detection System: After electrophoresis, the separated molecules need to be
visualized. For nucleic acids, this typically involves staining with fluorescent
dyes, followed by exposure to UV light. For proteins, methods such as
Coomassie Brilliant Blue staining, silver staining, or Western blotting can be
used to visualize the separated proteins.

Procedure for Slab Gel Electrophoresis

1. Preparation of the Gel:

The first step in slab gel electrophoresis is preparing the gel. If using agarose, the
agarose powder is dissolved in a buffer by heating, and then poured into a gel
casting tray with a comb inserted at one end to form the sample wells. The gel is left
to cool and solidify.

For polyacrylamide gels, a polymerization mixture containing acrylamide, bis-

acrylamide, and a polymerization initiator (such as ammonium persulfate) is
prepared. The mixture is poured into a gel casting tray, and the polymerization is
allowed to occur, forming a solid gel matrix.

2. Sample Preparation:

The biomolecule sample (e.g., DNA, RNA, or protein) is mixed with a sample buffer.
The sample buffer often contains glycerol to help the sample sink into the wells, and
SDS for protein samples to ensure denaturation and uniform charge distribution.
The samples are then loaded into the wells using a micropipette.

3. Running the Gel:

The gel is placed into the electrophoresis chamber, and an electrophoresis buffer is
added to the chamber to cover the gel. The power supply is connected, and an
electric field is applied. The voltage is adjusted depending on the size of the gel and
the sample. For nucleic acids, the electric current typically ranges from 50 to 150
volts, while for proteins, the current is usually higher.

As the electric field is applied, the charged molecules migrate through the gel matrix
at rates determined by their size and charge. Smaller molecules move faster than
larger ones.

4. Visualization:

After electrophoresis, the gel is removed from the apparatus, and the separated
molecules are visualized using appropriate methods. For DNA and RNA, the gel is
typically stained with a dye like ethidium bromide, which intercalates between the
bases of the nucleic acids and fluoresces under UV light. For proteins, various
staining techniques (e.g., Coomassie Brilliant Blue, silver staining) can be used to
visualize the proteins.

Applications of Slab Gel Electrophoresis

DNA Analysis: Agarose gel electrophoresis is commonly used to analyze the size of
DNA fragments, as well as to separate and purify DNA for further manipulation (e.g.,
cloning, sequencing).
Protein Separation: Polyacrylamide gel electrophoresis is used to separate proteins
based on their molecular weight, and SDS-PAGE is often used to denature proteins
and ensure size-based separation.

RNA Analysis: RNA molecules are separated by agarose gel electrophoresis, often to
check the integrity of RNA or to separate different RNA species (e.g., rRNA, mRNA).

5. Elaborate upon the applications of Agarose gel electrophoresis

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis (AGE) is a widely used technique in molecular biology

and biochemistry for the separation of nucleic acids (DNA and RNA) and proteins.
This method leverages the electric charge of the molecules and their differential
migration through an agarose gel matrix, which acts as a sieve. The applications of
agarose gel electrophoresis are vast and span multiple fields including genetic
research, clinical diagnostics, forensic science, biotechnology, and environmental
studies. Below is a detailed exploration of the various applications of agarose gel
electrophoresis.

1. DNA Analysis and Fragmentation

Agarose gel electrophoresis is most commonly used for the separation and analysis
of DNA fragments. It plays a crucial role in genetic research by allowing scientists to
analyze the size, purity, and quantity of DNA samples. Some common applications
include:

PCR Product Analysis: One of the most common uses of agarose gel
electrophoresis is to confirm the amplification of a target sequence via Polymerase
Chain Reaction (PCR). After amplification, PCR products are separated by size on
the gel to verify the success and specificity of the reaction.

Restriction Fragment Length Polymorphism (RFLP): Restriction enzymes cut DNA at

specific sequences. Agarose gel electrophoresis can separate these fragments,
enabling the analysis of genetic variations between different DNA samples.

DNA Fingerprinting: AGE is used in forensic science to compare DNA samples from
crime scenes with that of suspects. By separating DNA fragments produced by
restriction enzymes, a “fingerprint” can be obtained for comparison.
Gel Purification: After electrophoresis, specific DNA bands can be excised from the
gel, purified, and used for further applications such as cloning, sequencing, or
functional analysis.

2. RNA Analysis

Agarose gel electrophoresis is also valuable for the separation of RNA. The
technique helps determine the integrity of RNA samples, especially for high-quality
applications such as gene expression studies. Some important RNA-related
applications include:

RNA Integrity Check: In many studies, the integrity of RNA is critical for reliable
results. The 18S and 28S ribosomal RNA bands in the gel can be used as indicators
of RNA degradation. A distinct and sharp separation of these bands suggests high-
quality RNA, whereas degradation would cause smearing.

Northern Blotting: This is a technique used to detect specific RNA sequences after
separation by agarose gel electrophoresis. Northern blotting is often used in gene
expression studies and the detection of mRNA in various biological samples.

RNA Sequencing: Prior to RNA sequencing (RNA-Seq), agarose gel electrophoresis

is employed to confirm the size distribution and quality of RNA fragments. This step
is crucial for preparing RNA libraries for sequencing technologies.

3. Protein Analysis

While agarose gels are more commonly used for nucleic acids, they can also be
used for protein separation, though polyacrylamide gel electrophoresis (PAGE) is
typically preferred for proteins due to its higher resolution. However, agarose gel
electrophoresis still has valuable applications for certain proteins:

Isoelectric Focusing: Agarose gel electrophoresis can be used in combination with

isoelectric focusing (IEF), which separates proteins based on their isoelectric point
(pI). This technique is useful for analyzing complex protein mixtures and identifying
proteins with similar sizes but different charge properties.

Separation of Larger Proteins: Some large proteins, such as those involved in the
structure of chromosomes or cell membranes, can be separated more effectively in
agarose gels due to their less restrictive sieving properties compared to
polyacrylamide gels.
4. Genetic Mapping

Agarose gel electrophoresis is instrumental in the creation of genetic maps. By

separating DNA fragments derived from restriction enzyme digestion, it is possible
to determine the position of specific genes or markers in a genome. This is
particularly helpful in:

Linkage Mapping: Identifying the proximity of genes or markers on a chromosome

can be done by observing the co-segregation of DNA fragments during
electrophoresis. This helps in identifying genetic loci related to specific traits or
diseases.

Whole Genome Sequencing: Agarose gel electrophoresis is used to separate large

fragments of genomic DNA that are then mapped or sequenced for further analysis.
This is an essential step in understanding genome structure and function.

5. Cloning and Genetic Engineering

In molecular cloning, agarose gel electrophoresis helps identify recombinant DNA

molecules. For example, after restriction enzyme digestion and ligation into vectors,
the recombinant plasmids can be visualized by AGE. This ensures that only the
correct size and type of plasmid are used for further experiments such as
transformation into bacterial cells. Key applications in cloning include:

Confirmation of Ligation: After ligating DNA fragments into vectors, AGE helps
confirm whether the desired recombinant DNA has been successfully constructed
by analyzing the band patterns.

Screening of Clones: Agarose gel electrophoresis is used in the screening process of

bacterial colonies to determine which contain the recombinant plasmid, based on
the size of the insert.

6. Diagnosis of Genetic Disorders

In clinical diagnostics, agarose gel electrophoresis is used to detect genetic

mutations and to confirm the presence of specific genetic disorders. This is done by
identifying the pattern of DNA fragments associated with specific genetic
conditions, such as:

Cystic Fibrosis: Mutations in the CFTR gene can be detected using agarose gel
electrophoresis after PCR amplification of the gene, revealing deletion mutations.
 Sickle Cell Disease: AGE is used to differentiate between normal and sickle cell
hemoglobin by analyzing the size difference in the fragments produced by restriction
enzyme digestion of hemoglobin genes.

7. Environmental and Ecological Applications

Agarose gel electrophoresis is also used in environmental science to detect DNA

from various species in soil, water, or air samples, enabling studies in biodiversity
and ecosystem health. For example, environmental DNA (eDNA) can be extracted
from water samples and analyzed using AGE to detect the presence of specific
species without the need to capture or observe them directly. This application is
widely used in:

Biodiversity Monitoring: Detecting and monitoring endangered species or invasive

species in environmental samples.

Pollution Monitoring: Identifying microorganisms that degrade pollutants, by

analyzing microbial DNA in environmental samples.

6. Discuss various solubilizes and their role in characterization of

macromolecules

Solubilizers and Their Role in Characterization of Macromolecules

Macromolecules such as proteins, nucleic acids, polysaccharides, and synthetic polymers

are fundamental to a wide range of biological and industrial processes. However, due to
their large size, complex structure, and often hydrophobic or poorly soluble nature, their
characterization presents numerous challenges. One of the key strategies to overcome
these challenges involves the use of solubilizers. Solubilizers are agents that facilitate the
dissolution or stabilization of macromolecules in aqueous or organic solutions. Their role
in macromolecular characterization is crucial as they enable the study of these molecules’
physical, chemical, and biological properties.

1. Types of Solubilizers

Solubilizers can be broadly classified into two categories: surfactants and cosolvents.
Each type plays a unique role in macromolecular characterization.

Surfactants:

Surfactants are amphiphilic molecules that have both hydrophilic (water-attracting) and
hydrophobic (water-repelling) regions. These molecules are essential for solubilizing
hydrophobic macromolecules by forming micelles or other aggregates. Surfactants can be
anionic, cationic, nonionic, or zwitterionic, with each type having distinct effects on the
macromolecules they interact with.

Anionic surfactants (e.g., sodium dodecyl sulfate, SDS) are widely used to solubilize
proteins, especially for the analysis of their secondary and tertiary structures. SDS, for
example, denatures proteins by binding to their hydrophobic regions and inducing a
negative charge, allowing for their separation in electrophoresis.

Nonionic surfactants (e.g., Triton X-100, NP-40) are often used for protein extraction from
biological membranes without affecting the protein’s conformation, making them useful in
preserving protein function during characterization.

Zwitterion surfactants (e.g., CHAPS) are particularly useful when the preservation of
protein activity is important, as they can minimize protein denaturation while maintaining
solubility.

CoSolvents:

Cosolvents are organic or inorganic solvents that are added to water to enhance the
solubility of macromolecules. They can break the solvent-water structure, decrease
viscosity, or modify the solubility characteristics of macromolecules. Common cosolvents
include:

Alcohols (e.g., ethanol, glycerol) are used to reduce the hydrophobicity of certain
macromolecules, such as proteins, and facilitate their solubilization. Glycerol, for
instance, is frequently used to stabilize proteins and nucleic acids during storage.

Urea and guanidine hydrochloride are strong denaturants used in protein studies to unravel
and unfold the protein structure, enabling the characterization of folding and stability.

2. Role of Solubilizers in the Characterization of Macromolecules

Macromolecular characterization involves several techniques, including spectroscopy,

chromatography, electrophoresis, and microscopy. The solubilization of macromolecules
is essential for each of these methods, as solubilized macromolecules are more
accessible to analysis and can maintain their native structures.

Protein Characterization: In protein science, solubilizers are particularly important in

preventing protein aggregation and denaturation during experiments. For example, SDS is a
commonly used solubilizer in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), a technique used to analyze protein size and composition. The anionic nature
of SDS ensures that proteins are uniformly coated with a negative charge, allowing for their
separation based on molecular weight.
Solubilizers like urea and guanidine hydrochloride are also critical for understanding
protein folding and stability. These denaturants help researchers study the unfolding
pathways of proteins and identify regions crucial for their stability. Furthermore,
surfactants like CHAPS are used when studying membrane proteins, as they can solubilize
these proteins without causing the loss of their functional activity, which is essential for
understanding their structure and function.

Nucleic Acid Characterization: For nucleic acids, solubilizers like salt buffers or detergents
(e.g., Triton X-100) are used to prevent DNA or RNA from precipitating out of solution and to
break down cellular membranes during the extraction process. In techniques like gel
electrophoresis and spectrophotometry, solubilized nucleic acids can be analyzed for their
size, sequence, and structure. Cosolvents like glycerol are often added to preserve the
integrity of nucleic acids during storage, particularly during freeze-drying or long-term
storage.

Polysaccharide Characterization: Polysaccharides, being large, complex macromolecules,

often face solubility issues due to their hydrophobic and highly branched nature.
Solubilizers such as chaotropic agents (e.g., urea or guanidine) are frequently used to
disrupt the structure of polysaccharides, enhancing their solubility and making them
amenable to techniques like size-exclusion chromatography or nuclear magnetic
resonance (NMR) spectroscopy.

Synthetic Polymers: For synthetic polymers, solubilizers are often employed in techniques
like dynamic light scattering (DLS) or gel permeation chromatography (GPC) to measure the
size distribution and molecular weight of polymer samples. Polymers that are poorly
soluble in water may require organic solvents (e.g., tetrahydrofuran or dimethylformamide)
as cosolvents to aid in solubilization for analysis. Surfactants may also be used to modify
the surface characteristics of nanoparticles or polymer micelles for improved dispersion
and stability during characterization.

3. Impact of Solubilizers on Macromolecular Behavior

The choice of solubilizer can significantly influence the behavior of macromolecules during
characterization. For instance, using a surfactant like SDS can cause proteins to lose their
native structure, which might be beneficial for studying the denaturation process but is
detrimental for analyzing their functional state. In contrast, nonionic surfactants or low
concentrations of urea might preserve the secondary and tertiary structures, allowing for a
more accurate representation of the protein’s native state.

Moreover, solubilizers can affect the solubility and aggregation behavior of

macromolecules. For example, solubilizing proteins with high concentrations of salt can
sometimes induce aggregation, which would interfere with techniques like protein
crystallization. In such cases, using a mild detergent or cosolvent that stabilizes the protein
without inducing aggregation is preferred.

7. Give a narrative on sprotein characterization by SDS-PAGE

Protein Characterization by SDS-PAGE: A Narrative

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a fundamental

technique in biochemistry and molecular biology used to separate proteins based on their
molecular weight. It provides an essential tool for protein characterization, allowing
scientists to examine the purity, size, and relative abundance of proteins within complex
mixtures. This narrative provides a detailed overview of SDS-PAGE, from its principles and
methodology to its application in protein characterization.

Principles of SDS-PAGE

SDS-PAGE works on the principle of separating proteins by their size, which is determined
by the molecular weight. The technique utilizes two main components: sodium dodecyl
sulfate (SDS) and polyacrylamide gel. SDS is an anionic detergent that binds to proteins,
imparting a negative charge to them in a manner proportional to the length of the protein.
This means that the negative charge is effectively independent of the protein’s intrinsic
charge. By denaturing the proteins, SDS ensures that the protein structure is unfolded,
allowing the protein’s migration through the gel to be dictated solely by its size, rather than
its shape or charge.

The gel used in SDS-PAGE is made of polyacrylamide, a synthetic polymer. Polyacrylamide

forms a network of pores that acts as a molecular sieve. When an electric field is applied,
the negatively charged proteins migrate through the gel, with smaller proteins moving faster
through the pores and larger proteins encountering more resistance, hence migrating
slower. The resulting separation is based solely on the molecular size of the proteins, with
smaller proteins reaching the bottom of the gel more quickly than larger ones.

The SDS-PAGE Procedure

1. Sample Preparation: The first step in SDS-PAGE is the preparation of protein

samples. The proteins to be analyzed are usually extracted from biological samples
(e.g., tissues, cells, or culture media). The sample is then mixed with SDS-
containing buffer and heated to denature the proteins, ensuring they are uniformly
unfolded and coated with SDS.
 2. Loading the Gel: After preparation, the sample is loaded into the wells of the
polyacrylamide gel. The gel is typically cast between two glass plates, and the wells
are created at the top of the gel. Protein samples are mixed with a loading buffer that
contains SDS, a tracking dye, and often a reducing agent (such as dithiothreitol or β-
mercaptoethanol) to break any disulfide bonds between subunits. The tracking dye
enables monitoring of the electrophoresis process.
3. Electrophoresis: The gel is placed in an electrophoresis chamber and subjected to
an electric field. Proteins move through the gel towards the positive electrode, with
the rate of movement inversely proportional to their size. The electric field causes
the proteins to separate into bands, with each band representing proteins of similar
molecular weight.
4. Staining and Visualization: Once the electrophoresis run is complete, the gel is
stained to visualize the protein bands. The most commonly used stain is Coomassie
Brilliant Blue, which binds to proteins and allows their detection under visible light.
Other stains, such as silver staining, provide higher sensitivity for detecting lower-
abundance proteins. The protein bands can then be analyzed to estimate their size
and determine their relative abundance.
5. Molecular Weight Determination: A molecular weight marker (or protein ladder) is
often run alongside the samples. These markers consist of proteins of known
molecular weights and allow the determination of the size of the separated proteins
by comparison. The distance traveled by each protein is plotted against the
logarithm of its molecular weight, producing a standard curve. By comparing the
migration distance of unknown proteins to this curve, their molecular weights can
be estimated.

Applications of SDS-PAGE in Protein Characterization

SDS-PAGE is a versatile tool used in various aspects of protein characterization:

1. Protein Purity and Integrity: SDS-PAGE is commonly used to assess the purity of
proteins. In cases where the protein of interest is being purified, SDS-PAGE can
confirm whether the preparation contains contaminating proteins or other
impurities. The number of bands observed on the gel can reveal whether the protein
sample is homogeneous (one band) or heterogeneous (multiple bands). If there are
multiple bands, further purification may be necessary.
 2. Subunit Composition: For multi-subunit proteins, SDS-PAGE can be used to analyze
their subunit composition. When a protein is denatured, SDS will unfold it and
separate it into individual subunits. This provides information on the number of
subunits, their molecular weights, and whether they associate to form a functional
protein complex.
3. Estimating Molecular Weight: SDS-PAGE is widely used to estimate the molecular
weight of unknown proteins. By comparing the migration of the protein to known
molecular weight markers, scientists can approximate the size of the protein, which
is critical for understanding its structure, function, and potential post-translational
modifications.
4. Protein Expression Studies: SDS-PAGE is often employed to analyze the expression
levels of a particular protein. By comparing the intensity of the bands corresponding
to the protein of interest under different conditions (e.g., time points, experimental
treatments, or cell lines), researchers can assess changes in protein expression.
5. Detection of Post-Translational Modifications: While SDS-PAGE itself cannot directly
identify post-translational modifications, it can provide clues about modifications
that affect protein size, such as phosphorylation or glycosylation. A shift in the
protein’s migration pattern (e.g., a slower migration) may indicate the addition of a
modification, which can then be further investigated using other methods like mass
spectrometry or Western blotting.

Limitations and Alternatives

While SDS-PAGE is an invaluable tool for protein characterization, it has some limitations.
One major limitation is that SDS-PAGE cannot resolve proteins with very similar molecular
weights effectively. Additionally, while it can separate proteins based on size, it does not
provide information on their native structure, interactions, or functional activity. For these
purposes, techniques such as size-exclusion chromatography (SEC), mass spectrometry,
or native gel electrophoresis may be used in conjunction with SDS-PAGE.

Conclusion

SDS-PAGE remains one of the most widely used techniques in molecular biology and
biochemistry for protein characterization. It offers a powerful, reliable, and cost-effective
method for determining protein size, purity, and composition. By providing insights into the
molecular weight and structural aspects of proteins, SDS-PAGE continues to play a crucial
role in a variety of applications, from protein expression analysis to drug development.
Despite its limitations, SDS-PAGE is a cornerstone of proteomics and continues to be an
indispensable tool in the scientific community.
Short Answer Questions

1. Explain the role of buffers in electrophoresis.

Buffers play a crucial role in electrophoresis by maintaining a stable pH and providing ions
to conduct electricity through the gel medium. During electrophoresis, an electric field is
applied to move charged molecules, such as DNA or proteins, through a gel matrix. The
buffer system helps carry this current and ensures that the molecules retain their native or
denatured charges, depending on the technique used. The pH of the buffer must be
optimal to keep the biomolecules in a charged state, ensuring their proper migration based
on size or charge. In techniques like SDS-PAGE, a discontinuous buffer system is often
used to improve resolution by concentrating proteins into a thin band before separation.
Common buffers include TBE (Tris-borate-EDTA), TAE (Tris-acetate-EDTA) for nucleic acids,
and Tris-Glycine for proteins. The ionic strength of the buffer also influences the speed and
resolution of separation; low ionic strength results in slower migration but better
resolution, while high ionic strength speeds up migration but may cause overheating or
poor separation. Without a buffer, the electric current would not flow efficiently, and the pH
could fluctuate, leading to protein degradation or incorrect separation patterns.

2. Explain the chemistry of polymerization of acrylamide.

The polymerization of acrylamide is a chemical process used to create polyacrylamide

gels, commonly used in electrophoresis for the separation of proteins and nucleic acids.
Acrylamide (C3H5NO) is a monomer that, under the influence of a catalyst, polymerizes to
form long chains, resulting in a gel matrix. The process is typically initiated using
ammonium persulfate (APS) as a source of free radicals and N,N,N′,N′-
tetramethylethylenediamine (TEMED) as an accelerator. APS decomposes in the presence
of TEMED to produce free radicals, which then initiate the polymerization of acrylamide
molecules. To form a crosslinked gel, bisacrylamide (N,N′-methylenebisacrylamide) is
added. Bisacrylamide has two acrylamide groups that can crosslink with two different
acrylamide chains, forming a three-dimensional network. The concentration of acrylamide
and bisacrylamide determines the pore size of the gel, with higher concentrations resulting
in smaller pores suitable for separating smaller molecules. This polymerization must be
carefully controlled, as incomplete polymerization or incorrect ratios can lead to
inconsistent gel quality. The resulting polyacrylamide gel is chemically stable, transparent,
and provides a uniform medium through which molecules can migrate during
electrophoresis based on size, making it ideal for analytical and preparative separation
techniques.
 3. What detection methods are used to detect proteins in gel after electrophoresis?

After electrophoresis, several staining and detection methods are used to visualize and
analyze proteins in the gel. One of the most common methods is Coomassie Brilliant Blue
staining, which binds to proteins and produces a blue-colored band. It is sensitive,
relatively inexpensive, and suitable for detecting microgram levels of protein. Another
widely used technique is silver staining, which is significantly more sensitive than
Coomassie staining and can detect nanogram levels of protein. However, it requires more
steps and can be less consistent. Fluorescent dyes such as SYPRO Ruby or Deep Purple
offer high sensitivity and compatibility with imaging systems, making them useful for
quantitative analysis. Western blotting is another important method, where proteins are
transferred to a membrane after electrophoresis and detected using specific antibodies.
This technique allows for the identification of specific proteins within a complex mixture.
Additionally, autoradiography is used when proteins are radioactively labeled, producing an
image on photographic film. Each method varies in sensitivity, specificity, and ease of use.
The choice of detection method depends on the experimental goals, required sensitivity,
and available equipment, with more sensitive methods preferred for detecting low-
abundance proteins.

4. How can we determine subunit composition of proteins by SDS-PAGE?

SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) is an effective

method for determining the subunit composition of proteins. SDS is an anionic detergent
that denatures proteins by disrupting non-covalent bonds and imparts a uniform negative
charge proportional to the length of the polypeptide chain. In the presence of SDS and a
reducing agent like β-mercaptoethanol or dithiothreitol (DTT), protein complexes are
dissociated into their individual subunits by breaking disulfide bonds. During
electrophoresis, these denatured and uniformly negatively charged polypeptides migrate
through a polyacrylamide gel matrix based on their molecular weight. After
electrophoresis, the gel is stained to visualize the protein bands. The number of bands
corresponds to the number of distinct subunits, and their relative positions indicate the
molecular weights. By comparing the positions of the protein bands with those of
molecular weight standards, the size of each subunit can be determined. This approach
helps in understanding the quaternary structure of proteins and identifying whether a
protein is composed of multiple identical or different subunits. Therefore, SDS-PAGE is a
powerful analytical tool for protein characterization, especially when studying oligomeric
proteins and their subunit composition.
 5. Write a short note on submarine gel electrophoresis.

Submarine gel electrophoresis is a commonly used technique for the separation and
analysis of nucleic acids, particularly DNA and RNA. The term “submarine” refers to the gel
being submerged in buffer during electrophoresis, typically using an agarose gel slab. The
gel is cast horizontally in a tray with wells formed at one end to load samples. Once
solidified, the gel is placed in an electrophoresis chamber filled with buffer (usually TAE or
TBE), completely covering the gel, which helps in uniform current flow. DNA samples mixed
with a loading dye are loaded into the wells, and an electric field is applied, causing
negatively charged DNA molecules to migrate toward the positive electrode. The migration
rate depends on the size of the DNA fragments, with smaller fragments moving faster
through the gel matrix. After electrophoresis, the gel is stained with a DNA-binding dye like
ethidium bromide or SYBR Green, and the bands are visualized under UV light. Submarine
gel electrophoresis is widely used in molecular biology for applications like checking the
quality of DNA/RNA, assessing PCR products, and estimating DNA fragment sizes by
comparing with molecular markers.

6. Write a short note on molecular weight analysis by electrophoresis.

Electrophoresis is a powerful tool for determining the molecular weight of biomolecules

such as proteins and nucleic acids. For proteins, SDS-PAGE is commonly used. In this
method, SDS denatures the proteins and gives them a uniform negative charge, allowing
them to be separated solely based on size. During electrophoresis, proteins migrate
through the polyacrylamide gel matrix, and smaller proteins move faster. By comparing the
migration distances of unknown proteins to a set of molecular weight standards (also
known as protein ladders), the molecular weights of the sample proteins can be estimated.
Similarly, agarose gel electrophoresis is used for nucleic acids, where DNA or RNA
fragments are separated by size. A DNA ladder containing fragments of known sizes is run
alongside the samples for comparison. The distance migrated by each fragment is inversely
related to its size, and a standard curve (log of molecular weight vs. migration distance) can
be plotted to determine the size of unknown fragments. These methods are widely used in
research and diagnostics to characterize biomolecules, confirm the success of genetic
modifications, or detect mutations. Accurate molecular weight determination is crucial for
protein identification and nucleic acid analysis.

7. Explain the role of discontinuous buffer system in SDS-PAGE.

A discontinuous buffer system is essential in SDS-PAGE for improving resolution and

separating proteins more effectively. It uses different buffer compositions in the stacking
gel, resolving (separating) gel, and electrode buffers. Typically, the stacking gel has a low
pH (~6.8) and lower acrylamide concentration, while the resolving gel has a higher pH
(~8.8) and higher acrylamide concentration. The electrode buffer (commonly Tris-glycine)
maintains a constant pH and ionic environment. When electrophoresis begins, the
proteins, SDS, and buffer ions form moving boundaries due to their different
electrophoretic mobilities. In the stacking gel, glycine is largely uncharged and migrates
slowly, while chloride ions move rapidly, creating a voltage gradient. Proteins are
sandwiched between these ion fronts and get compressed into thin, sharp bands — a
process called “stacking.” This concentration effect ensures that all proteins start migrating
into the resolving gel simultaneously, allowing for high-resolution separation based on
molecular weight. As the proteins enter the resolving gel, glycine becomes more negatively
charged and overtakes the proteins, and separation occurs. This system dramatically
improves the clarity and resolution of protein bands, making it a critical component of SDS-
PAGE methodology.

8. Write a short note on recovery of molecules from the gel.

Recovery of molecules from the gel, also known as gel extraction or gel elution, is a process
used to isolate and purify specific DNA, RNA, or protein fragments after electrophoresis.
Once the desired band is visualized under UV light or with staining, it is carefully excised
from the gel using a sterile scalpel or blade. For nucleic acids, the gel slice is then
processed using commercial gel extraction kits or traditional methods. These often involve
dissolving the agarose in a chaotropic salt solution, binding the nucleic acid to a silica
membrane, washing away impurities, and eluting the purified DNA or RNA. In the case of
proteins, polyacrylamide gels require more complex methods such as electroelution,
where an electric field is applied to pull the protein out of the gel into a buffer chamber, or
passive diffusion, though less efficient. Gel recovery is commonly used in molecular
biology for cloning, sequencing, PCR, or further biochemical analysis. It enables the
purification of a specific fragment from a mixture, ensuring accuracy in downstream
applications. Care must be taken to minimize contamination and degradation during the
extraction process to maintain sample integrity.
Very Short Answer Questions:

1. What is the role of electrolysis during electrophoresis?

It generates the electric field that drives the movement of charged molecules.

2. What happens when nucleic acids are treated with urea?

Urea denatures nucleic acids by disrupting hydrogen bonds.

3. Nucleic acids migrate towards which electrode during electrophoresis?

They migrate towards the positive electrode (anode).

4. Name the gel with least molecular sieving ability.

Agarose (especially at low concentrations).

5. What is reptation?

Snake-like movement of linear DNA through a gel matrix.

6. Which compound is used to activate polymerization of acrylamide monomers?

Ammonium persulfate (APS).

7. Name an alkylating agent used during electrophoresis.

Ethidium bromide.

8. What is used to detect nucleic acids after electrophoresis?

Ethidium bromide or SYBR Green.

9. Which molecular parameter can be leveraged to separate proteins with same

charge/mass ratio?

Shape or conformation.

10. Name a cationic detergent used as solubilizer.

Cetyltrimethylammonium bromide (CTAB).