This document discusses gel electrophoresis, which separates biomolecules like DNA, RNA, and proteins based on their size and charge. It describes the basic principles of electrophoresis, including how mobility depends on factors like net charge and size. The key steps of gel electrophoresis are explained, such as preparing the gel, loading samples, running the current, and staining. Two-dimensional gel electrophoresis is also summarized, which separates proteins based on isoelectric focusing in the first dimension and size in the second dimension, allowing high resolution separation of thousands of proteins.

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SUMBUL
M.PHIL 2ND SEMISTER
PRESENTATION TOPIC: GEL ELECTROPHORESIS
UNIVERSITY OF KARACHI

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CONTENTS
TOPIC PAGE NO.
GEL ELECTROPHORESIS 3
i. Introduction 3
ii. History of electrophoresis 3
iii. Principle of electrophoresis 3
iv. Mobility or Rate of Migration 4
v. Apparatus and working of gel electrophoresis 5
vi. Media for Electrophoresis 6
vii. Staining of gel 8
viii. Two dimensional electrophoresis 8
ix. Principle of 2-D electrophoresis 9
x. Process of 2-D gel electrophoresis 9
xi. Isoelectric point (pi) and isoelectric focusing (IEF) 10

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GEL ELECTROPHORESIS
Introduction:
Electrophoresis: The prefix Electro means flow of electron or current. While the suffix Phoresis mean
movement. Thus Electrophoresis is movement underelectric current and Gel electrophoresis is therefore a technique
used in the lab to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size
and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on
their size and charge, the molecules will travel through the gel in different directions or at different speeds,allowing
them to be separated from one another. Gel electrophoresis has a variety of applications; for example, it is used in
DNA fingerprinting and the detection of genetic variants and proteins involved in health and disease as well as in the
detection and purification of nucleic acids and proteins for research.It is also used to aid in the detection of pathogens
(disease-causing organisms)that may be present in blood or othertissues orin sources such as food.In many instances,
nucleic acids or proteins that are detected and purified with gel electrophoresis are investigated further by means of
DNA sequencing or mass spectrometry.
History of electrophoresis: The history of electrophoresis for molecular separation and
chemical analysis began in 1931 with the work of Arne Tiselius known as Father of
Electrophoresis, while new separation processes and chemical analysis techniques based
on electrophoresis continue to be developed in the 21st century.
Principle of electrophoresis:
If a mixture of electrically charged biomolecules is placed in an electric field of field strength E, they will
freely move towards the electrode of opposite charge. However, different molecules will move at quite different and
individual rates depending on the physical characteristics of the molecule and on experimental system used. The
velocity of movement, ν, of a charged molecule in an electric field depends on variables described by
  E.q/ f
• Where v = velocity of migration of the molecule.
• E = electric field in volts per cm
• q = net electric charge on the molecule

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• f = frictional coefficient
The movement of charged particle in an electric field is
Expressed in terms of electrophoretic mobility, denoted by µ.
Where,
µ = v/E OR µ = q/f
For molecules with similar conformation f varies with size but not with shape. Electrophoretic mobility (µ) of a
molecule is directly proportional to chargemass ratio.
The frictional coefficient describes frictional resistance to mobility and depends on a number of factors such as mass
of the molecule, its degree of compactness, buffer viscosity and the porosity of the matrix in which the experiment is
performed. The net charge is determined by the number of positive and negative charges in the molecule. Charges are
conferred on proteins by amino acid side chains as well as by groups arising from post translational modifications
such as deamination, acylation or phosphorylation. DNA has a particularly uniform charge distribution since a
phosphate group confers a single negative charge per nucleotide. Equation 1 means that, in general molecules will
move faster as their net charge increases, the electric field strengthens and as f decreases (which is a function of
molecular mass/shape). Molecules of similar net charge separate due to differences in frictional coefficient while
molecules of similar mass/shape may differ widely from each other in net charge. Consequently,it is often possible
to achieve very high resolution separation by electrophoresis.
Charges molecules: Charge in DNA, RNA and Nucleotides is due to Phosphate groups in these molecules Proteins
and Amino acids is due to presence of ionizable group e.g. amino, carboxylic. Hence these molecules are subject to
separation by electrophoresis
Mobility or Rate of Migration:
Separation can be effected by either or both major components. The mobility or rate of migration of ions in
electrophoresis is dependent upon the following factors:
 Net charge of the molecules
 Size and shape of the molecules
 Support medium properties

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 Strength of the electrical field
 Ionic strength of the buffer
 Temperature
Electrophoresis is not always run to endpoint- if molecules are detected in matrix, empirical endpoint chosen such that
all molecules still in matrix.
 Charge – higher the charge greater the electrophoretic mobility.
 Size – bigger the molecule greater are the frictional forces exerted on it by the medium. Larger particles have
smaller electrophoretic mobility compared to smaller particles.
 Shape – rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours.
Therefore globular protein move faster than fibrous protein.
Apparatus and Working of Gel Electrophoresis:
The gel electrophoresis apparatus consists of
i. Electrophoresis chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end
and an anode (positive terminal) at the opposite end.
ii. Agarose gel
iii. Gel casting tray
iv. Buffer
v. Staining agent (dye)
vi. A comb
vii. Ladder
viii. Sample to be separate
The gel is prepared between the two sandwiched glass plates.Initially, solution for separation gel is poured in between
the glass plates, over which stalking gel is layered. Comb is inserted immediately to cast wells. The polyacrylamide
gel is removed and placed in the buffer filled separating tank. Samples are mixed with tracking dye bromophenol blue
and sucrose.The mixed sample is then loaded (100 µl) in the wells along with markers. The power pack with an output

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up to 500 volts is switched on to supply direct current. After electrophoresis, gel has to be treated with suitable dye
which produces stable colored compound after binding with DNA or proteins. The gel, which contains a series of
wells at the cathode end,is placed inside the chamber and covered with a buffer solution.The samples are then loaded
into the wells with a pipette. The chamber is connected to a power supply that, when turned on, applies an electric
field to the buffer. The electric field causes negatively charged molecules to migrate through the gel toward the anode.
(DNA and RNA are negatively charged; proteins must be treated with a detergent to give them a negative charge.)
The molecules’ movement is influenced by the porous gel matrix such that larger, heavier molecules move relatively
slowly, whereas smaller, lighter molecules move more quickly. The density of pores and the type of substance used
to make the gel further influence the rate of molecule migration. Often a dyed “ladder,” or marker with multiple
molecules of known and varying molecular weights, is run alongside experimental samples to serve as a reference for
size. The dye enables the visualization of the marker as it moves through the gel; samples typically are also dyed for
visualization.
Media for Electrophoresis:
In general the macromolecules solution is electrophoresed through some kind of matrix. The matrix acts as a molecular
sieve to aid in the separation of molecules on the basis of size. The kind of supporting matrix used depends on the
type of molecules to be separated and on the desired basis for separation: charge, molecular weight or both (Dolnik,
V.; 1997). The most commonly used materials for the separation of nucleic acids and proteins are agarose and
acrylamide
Polyacrylamide Gel Electrophoresis (PAGE):

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Polyacrylamide is commonly known as PAGE is widely used for separation of proteins and low molecular weight
DNA fragments. Polyacrylamide is the choice of gel matrices in PAGE process. Polyacrylamide is formed by the
polymerization of acrylamide and N, N’ methylene-bis-acrylamide. Bis-acrylamide acts as cross-linking agent. Such
gels are suitable for high-resolution separation of DNA and proteins across a large mass range.
• Polymerization process is triggered by the addition of ammonium persulphate and tetramethylethylene
diamine (TEMED). Formation of free radicals from persulphate ions is aided by TEMED. PAG contains SDS
– an anionic detergent, disrupts nearly all non-covalent interactions
– Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding
protein.
– Binds to protein in a ratio of one SDS molecule/two
amino acids
– Masks’ charge on protein so that all proteins are
uniformly negatively charged
– Prevents protein aggregation
– Prevents protein shape frominfluencing gel run
SDS-PAGE, sodiumdodecyl sulfate polyacrylamide gel electrophoresis,
describes a collection of related techniques to separate proteins according to their electrophoretic mobility (a function
of the molecular weight of a polypeptide chain) while in the denatured (unfolded) state.In most proteins, the binding

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of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a
fractionation by approximate size during electrophoresis.
SDS is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides.When a protein
mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. In this process,
the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS.
Thus polypeptides after treatment become rod-like structures possessing a uniform charge density that is same net
negative charge per unit length. The electrophoretic mobility’s of these proteins will be a linear function of the
logarithms of their molecular weights.
Staining Of Gel: One of the most important aspects of gel
electrophoresis technique is staining. Once sample molecules
have separated in the gelmatrix it is necessary to visualize their
position.This is achieved by staining with an agent appropriate
for the sample. Some of the more common staining methods
used in biochemistry are listed below:
These limits of detection should be regarded as approximate
since individual protein may stain more or less intensely then
average. Commonly used stain for biopolymers after
electrophoretic separation in agarose or polyacrylamide gels
Two Dimensional Electrophoresis:
Two-dimensional gel electrophoresis (2DE) has been a
mainstay of proteomic techniques for more than four decades.
Only “Proteomics” is the large-scale screening of the proteins
of a cell, organismor biological fluid, a process which requires
stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot
STAIN USE DETECTION
LIMIT a (ng)
Amido black Proteins 400
Coomassie Proteins 200
Ponceau red Proteins
(reversible)
200
Bis-1-anilino-8-
Naphthalene sulphonate
Proteins 150
Nile red Proteins
(reversible)
20
SYPRO orange Proteins 10
Fluorescamine (protein
treated prior to
electrophoresis)
Proteins 1
Silver chloride Proteins/DNA 1
SYPRO red Proteins 0.5
Ethidium bromide DNA/RNA 10

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identification, and database searches. The core technology of proteomics is 2-DE.At present, there is no other
technique that is capable of simultaneously resolving thousands of proteins in one separation procedure.
The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or
even thousands of proteins simultaneously.
Principle Of 2-D Electrophoresis:
Some years ago, the basic protocol for 2-DE was described. This protocol is still valid today: isoelectric focusing
(IEF) is performed and after equilibration with SDS buffer in
the presence of DTT, the IEF gel is applied to a SDS gel
which allows the resolution of hundreds oreven thousandsof
proteins present in a particular cell, while allowing synthesis
rates to be determined quantitatively, all in one experiment.
The procedure has become the core technology to visualize
the global changes in protein expression for proteome
analysis, permitting higher resolution, improved
reproducibility, higher loading capacity, and simplicity for
preparative 2-DE with subsequent spot identification by
mass spectrometry.
Process of 2-D gel electrophoresis:
Two-dimensional gel electrophoresis (2DGel) is a successfulmethod used for the detection and analysis of proteins.
It has been designed as a combination of the 2DGel, IEF and SDS-PAGE methods, and is used in the analysis of
complex protein mixtures. In the first step,protein is separated into its charges with IEF, whereas in the second step,
the protein is separated according to its mass. The separated protein on the gel with IEF is negatively charged by
treatment with SDS, and the electrophoresis is performed by inserting the gel horizontally into the SDS-PAGE gel.
(Fig. 15.4). Thus,the proteins that are focused on the pI are separated according to their molecular weights. Generally
20×20 cm large gels are used in SDS-PAGE setup and more than 10,000 proteins can be separated. If the protein
amount is around 10 ng, Coomassie dye is used and if the protein amount is around 0.5 ng, silver or fluorescent total-

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protein satins can be used for detection. Using the systemknown as “ISO-DALT,” both IEF and SDS-PAGE can be
carried out simultaneously (Chen et al., 2015; Brunelle and Green, 2014; Hanash et al., 1991; Magdeldin et al., 2014).
Isoelectric Point (Pi) And Isoelectric Focusing (IEF):
Proteins carry both basic and acidic side chains. Basic side chains ionize in acidic pH and carry positive charge, and
vice versa, so at low pH proteins are more positively charged, at high pH proteins are more negatively charged. At a
certain pH the proteins have equal positive and negative charges, such a pH is called an ISOELECTRIC POINT.
The first dimension in a 2-D gel electrophoresis
experiment involves the separation of proteins
according to their isoelectric point (pI) by isoelectric
focusing (IEF). IEF works by applying an electric field to protein within a pH gradient. The proteins separate as they
migrate through the pH gradient in response to the applied voltage. When a protein reaches a pH value that matches
its pI, its net electrical charge becomes neutral, and stops migrating. In this way, each protein in a sample becomes
"focused" according to its pI. IEF can be performed using two techniques: immobilized pH gradients (IPG) with
ampholytes covalently bound to a gel, or carrier ampholytes that migrate through a gel to generate the pH gradient.
Proteins will move in an electric field only when they are at a pH other than their pI, the pI of cytochrome c, a highly
basic protein is 10.6 and of serum albumin, a basic protein is 4.8.
References:
 Techniques for Protein Analysis by Gülay Büyükköroğlu, Candan Hızel, in Omics Technologies and Bio-
Engineering, 2018.
 Protein Electrophoresis Handbook.
 Isoelectric Focusing in 2D Electrophoresis by bio-rad.
 Gel electrophoresis - advanced techniques by Sameh Magdelin
 2-D Electrophoresis Principles and Methods TomBerkelman and Tirra Stenstedt with contributions from
Bengt Bjellqvist Nancy Laird Michael McDowell Ingmar Olsson Reiner Westermeier.