ELECTROPHORESI

S
CONTENTS

INTRODUCTION
PRINCIPLE
PROCESS INVOLVED IN
ELECTROPHORESIS
INSTRUMENT OVERVIEW
TYPES OF ELECTROPHORESIS
APPLICATION
USES OF VARIOUS SCIENCES
 INTRODUCTION
 Electrophoresis consists of two words; electro meaning electricity, and phoresis meaning
movement. An electrophoretic system consists of two electrodes of opposite charge (anode,
cathode), connected by a conducting medium called an electrolyte.
 Thus, it implies the migration and separation of a charged particle (ions) through a
solution under the influence of an electric field.
 When an electric field is applied across two electrodes that are totally submerged in a
colloidal solution, the particles (colloidal) tend to move towards one or the other electrode.
This movement of particles under the effect of the electric field is known as electrophoresis.
 How do we find the existence of charge on colloidal particles? Electrophoresis is the
answer.
 PRINCIPLE BEHIND
ELECTROPHORESIS
 When charged molecules are placed in an electric field, they migrate toward either the
positive or negative pole according to their charge. In contrast to proteins, which can have
either a net positive or net negative charge, nucleic acids have a consistent negative charge
imparted by their phosphate backbone, and migrate toward the anode.
 An ion placed in such an electric field will experience a force
This force will cause the protein to accelerate towards either the cathode or the anode,
depending on the sign of its charge. Of course there are other forces such as frictional force
when ions move in the electric field.
HORIZONTAL ELECTROPHORESIS
UNIT
 PROCESS INVOLVED IN
ELECTROPHORESIS

a) SEPERATION
b) DETECTION

c) QUANTIFICATION
 SEPERATION

 The instrument set up is according to its type. In the gel

electrophoresis, gels are prepared and cast. Then placed into the
electrophoresis chamber. The supportive medium can be agarose
gels or polyacrylamide gels. Then appropriate buffer solution is
added to the system. After the proper setup of the instrument, the
sample is placed into the medium. Then the sample is run at a
specific current, voltage, or power.
 DETECTION

 Staining with a dye or autoradiography (for radioactive samples)

helps in the detection of the separated components.
 QUANTIFICATION

 Quantification is done using a densitometer or by direct

measurement using an optical detection system. For example,
protein is fixed by precipitating in gel with acetic acid. Methanol
helps prevent the diffusion of proteins from the gel during the
staining process.
 INSTRUMENT OVERVIEW
Modern electrophoresis equipment and systems vary based on its types and forms. However, all
the electrophoretic system possesses two essential components :
1. Power pack
 Power supply drives the movement of ionic species in the medium and allows adjustment and
control of either the current or the voltage.

2. An electrophoresis unit
 An electrophoretic system depends on its type but essentially consists of two electrodes of
opposite charge (anode and cathode), connected by a conducting medium called an
electrolyte. In addition, a supportive medium is present in electrophoretic systems like gel and
paper electrophoresis.
 TO BE CONTD

Buffer (Electrolyte)
 Buffers carry applied electric current and provide appropriate pH for the process.
Conducting (running) buffers like Tris borate EDTA (TBE) and Tris-acetate acid
EDTA (TAE) are commonly used.
Supportive Medium
 The supportive medium is the matrix (gel), in which biomolecules are separated. It
can be in the slab or capillary form. The supportive mediums used are sugar
polymers like agarose gel, polyacrylamide gel, starch gel, and cellulose acetate gel.
The medium runs either vertical or horizontal gel systems in gel electrophoresis.
Horizontal: agarose gel electrophoresis, and vertical: SDS-PAGE. The higher the
pore size, the higher the speed traveled by charged particles.
TYPES OF ELECTROPHORESIS

Capillary electrophoresis
1. Paper electrophoresis
2. Gel electrophoresis
PAPER ELECTROPHORESIS
INTRODUCTION

Paper electrophoresis employs filter paper strips soaked in buffer solution,

usually diethylbarbituric acid and barbituric acid dissolved in alkali (Veronal
buffer), pH 8.6. A small volume of serum is placed on the paper and a direct
current passed for several hours.
 PROCEDURE
The sample may be applied as a spot (about 0.5cm in diameter) or as a narrow uniform streak.
Special devices are available commercially for this purpose.
The sample can be applied before the paper has been equilibrated with buffer (or) after it.
After the sample has been applied to the paper and the paper has been equilibrated with the
buffer.
The current is switched on.
Commonly used buffers are , BARBITAL,PHOSPHATE , ACETATE , CITRATE , MICHALIES
The device providing stable voltage (or) current is available.

Frequent observation is necessary to run electrophoretic apparatus.

Overheating can be avoided by placing the entire equipment in the cold room.

The process does not take longer than two hours.

After 2 hours switched off the power and paper is removed.

Once removed, the paper is dried in hot oven at 1100C.

GEL ELECTROPHORESIS
INTRODUCTION
 Gel electrophoresis is simple, rapid and sensitive analytical technique for the separation
of charged particle.
 The gels, however, are porous and the size of the pores relative to that of the molecule
determines whether the molecule will enter the pore and be retarded or will bypass it.
The separation thus not only depends on the charge on the molecule but also on its size.
Needless to say, that resolution of a sample is sharper and better in a gel than in any
other type of medium.
 Agrose gel is used as a supporting media for the separation of DNA, RNA or protein
under the influence of electric charge.
 Most of the biomolecules has a net charge at any pH other than at their isoelectric
point.
 There is difference in the electrophoretic mobility of these charged molecules due to
their difference in size, shape, and charge.
There are basically two types of materials
are used to make gels:

 Agarose
 Polyacrylamide
 AGAROSE
 Agarose is natural colloid which is isolated from the
seaweed.
 It is linear polysaccharide.
 It is made up of repeating units of agarobiose, comprises
alternating units of 3,6-anhydrolactose and galactose.
 This gel has generally larger pore size, which makes them
suitable to separate larger molecules having molecular mass
more than 200 kDa.
 It is most commonly used for the electrophoresis of both
protein and nucleic acids.
 Agarose is used in concentration between 1% and 3%.
POLYACRYLAMIDE
 Polyacrylamide gel is consisting of chains of acrylamide
monomers crosslinked with N, N’-methylenebisacrylamide
units, which is commonly termed as bisacrylamide.
 In this gel, pore size and resolving power is totally depends
upon the concentration of acrylamide and bisacrylamide.
 The concentration of the gel normally varies from 5% to
25%.
 This gel is used in electrophoresis for the separation of
proteins ranging from molecular weight <5000 to
>200,000, and polynucleotides ranges from <5 to ~ 3000
base pairs in size.
APPARATUS OF GEL ELECTROPHORESIS

 Vertical gel apparatus: It is commonly used in sds PAGE for the separation of proteins.

 Horizontal gel apparatus: It is used for immune electrophoresis, iso-electric focusing and
electrophoresis of DNA and RNA in the agarose gel.
 Horizontal Gel Electrophoresis Vertical Gel Electrophoresis
Description
It is a technique where the gel is cast It is a technique where the gel is cast in a vertical
horizontally. direction.

Nature of Buffer
Continuous buffer is used in horizontal gel It uses a discontinuous buffer.
electrophoresis.

Nature of Gel
Horizontal gels are mostly made of agarose. Vertical gels are mostly made of acrylamide.

Macromolecule Separated
It is used to separate nucleic acids such as RNA It is used in separating proteins.
and DNA.

Position of Anode and Cathode

Anode and cathode are present at either ends of Cathode and anode are present at the top and
the electrophoretic chamber. bottom of the chamber.
TYPES OF GEL ELECTROPHORESIS

 AGAROSE GEL ELECTROPHORESIS

 SDS-PAGE
 PULSE FIELD GEL ELECTROPHORESIS
(PFGE)
 2D GEL ELECTROPHORESIS
 1. AGAROSE GEL ELECTROPHORESIS

 Agarose gel the supporting media in the electrophoresis.

 For the electrophoresis of DNA, RNA and Protein agarose gel is used.
 PRINCIPLE
 In the agarose gel electrophoresis the potential difference is applied across the electrodes in a horizontal
electrophoretic tank containing agarose gel and biomolecules (such as nucleic acid or proteins) is
loaded, then molecules migrated to their respective electrodes. The rate of migration of charged
particles depends on the size, shape, molecular mass etc.

 In this process, larger molecules have difficulty in moving through the pore size of the supporting
media, whereas the smaller molecules has more mobility through it. The bands of protein or nucleic
acid is visualized by using intercalating dye, i.e., ethidium bromide (Etbr), they are visualized by
fluorescence when illuminated with ultraviolet lights.
 ELECTROPHORESIS BUFFER
 Composition and ionic strength of electrophoresis buffer is most important factor for the separation of
nucleic acids (DNA or RNA).

 Most routinely used buffers are: TAE- (Tris-acetate-EDTA), it has lower buffering capacity and
generally used to separate larger nucleic acid fragments (>12kb). TBE- (Tris-borate-EDTA), it has high
buffering capacity and higher ionic strength and generally used for the separation of low molecular
weight compound (<1kb). Loading buffer: Nucleic acid is before loading on to a gel is first mixed with
the gel loading buffer, which usually consists of:- Salts: It creates environment with favorable ionic
strength and pH of the sample, e.g., Tris-HCl. Metal chelator: It prevents nucleases to degrade the
nucleic acid such as EDTA. Loading dyes: It provides color for tracking and easy monitoring of sample.
Such as, bromophenol blue, xylene cyanol.Transilluminator: (An ultraviolet light box), which is used to
visualize bands in gels.
2. SDS – PAGE ELECTROPHORESIS
 Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis is routinely used for the
separation of proteins on the basis of their mass.
 It involves the use of vertical gel apparatus to separate proteins.
Requirement/ Instrumentation
 Gel or media acrylamide solutions.
 Buffer system the separation and migration patterns of proteins in gel
electrophoresis are determined by the chemical composition and pH of the
buffer system.
Three basic types of buffers are required:
 Gel casting buffer to cast gel
 Sample buffer to prepare the sample
 Running buffer to fill the electrode reservoirs
 lower reservoir amine buffer with HCl (running gel) Upper reservoir amine buffer with glycine
(stack gel) Staining or tracking dye
 Protein samples
 Molecular weight markers
 An electrophoresis chamber and power supply
 Glass plates (a short and a top plate)
 Casting frame
 Casting stand
 Combs
 3. PULSE FIELD GEL
ELECTROPHORESIS [PFGE]
 Conventional agarose gel electrophoresis cannot separate linear double stranded DNA molecules that
have radius of gyration, which is larger than the pore size of the gel.
 However, with certain changes in the orientation of electric field with respect to the gel, large DNA
fragments can be resolved.
 It is used to separate large DNA molecule by applying gel matrix as electric field that periodically
changes direction.
 This technique is invented by Schwartz and Cantor in 1984.
 DNA fragments up to 10 mb can be separated by this technique.
 PRINCIPLE
 As opposed to the continuous unidirectional electric fields applied in conventional gel electrophoresis,
pulsed-field gel electrophoresis uses pulsed, alternating, orthogonal electric fields. When such a field is
applied to a gel, large DNA molecules become trapped into their reptation tubes every time the direction
of the electric field is changed.
 These molecules remain immobile till they reorient t themselves along the direction of the new electric
field. It is here that different DNA molecules adapt a behaviour consonant with their respective sizes; ;
large DNA molecules take a longer time to reorient themselves and are consequently retarded more in
the new electric field as compared to the smaller DNA molecules.
 TO BE CONTD
 Thus, all those molecules of DNA whose reorientation times are less than the period of the electric pulse
can be fractionated in a size dependent manner.

 Factors, which are of extreme importance for determining the limit of resolution of pulsed-field gel
electrophoresis are given below:

 The absolute periods of the electric pulses.

 The angles at which the two electric fields are applied to the gel.
 The relative field strengths of the two electric fields and the degree of uniformity of the two electric
fields.
 The ratio of the periods of the electric pulses employed to generate the two electric fields.
 INSTRUMENTATION

The original apparatus used pulsed electric fields or

perpendicular orientations and linear electrodes.
 4. 2D GEL ELECTROPHORESIS
 Analysis of sample by one-dimensional electrophoresis is the most common form
of protein gel electrophoresis.
 For separation and analysis of hundreds to thousands of proteins in one gel, a
powerful electrophoretic method called two-dimensional gel electrophoresis is
used.
 2D gel electrophoresis separates a mixture of proteins according to two
properties, one in each dimension.
 The first dimensions involve the separation based on native isoelectric point (pI),
using form of electrophoresis called isoelectric focusing (IEF).
 Second dimensions separate mass using SDS-PAGE.
 This technique provides highest resolution for the protein analysis.
 APPLICATION OF
ELECTROPHORESIS
It is applied for routine laboratory experiments, disease diagnosis, research-oriented separations
and identification. Similarly, it is used in various other fields, like forensics, agriculture,
pharmaceutical, foods, etc.

1] DNA Analysis and DNA Fragmentation

Gel electrophoresis is the core technique for genetic analysis and purification of nucleic acids
for further studies or disease diagnosis.
2] Identifying Specific protein
 The rate of movement of macromolecules in an electric field is a helpful parameter to know
any changes in amino acids regarding their charge.
 Quantitative analysis of specific serum protein classes such as gamma globulins and
albumins
 It helps in the identification and quantitation of hemoglobin and its subclasses.
 It also helps in the identification of monoclonal protein in either serum or urine.
 Likewise, it helps in the separation and quantitation of significant lipoprotein classes.
 Immuno electrophoresis helps to analyse several kinds of protein’s existence and how they
behave chemically in different environments.
 It is also helpful in purifying proteins for different purposes.
 Similarly, it is useful in determining the molecular weights of protein.
3] Coenzymes separation
It is useful in separating and quantifying coenzymes such as creatine, kinase, lactate
dehydrogenase, and alkaline phosphatase coenzyme to their respective subtypes.

4] analysis of chemical compounds

 It helps analyse compounds, such as water, soil, air quality or contamination, food quality,
processing hygiene, and medical forensic analysis.
 It also helps in analysing transition metals.
 Likewise, it helps to analyse organic compounds.
 Similarly, it helps in analysing components of pesticides.
 ELECTROPHORESIS USED IN VARIOUS SCIENCES
LIFE SCIENCE
Using electrophoresis, we can
see how many different DNA
fragments are present in a
sample and how large they are
relative to one another. We can
also determine the absolute
size of a piece of DNA by
examining it next to a standard
"yardstick" made up of DNA
fragments of known sizes.
BIOLOGICAL SCIENCE
Electrophoresis is an
essential technology for the
separation and analysis of
nucleic acids.
Electrophoresis of nucleic
acids is used routinely at
the lab bench for the
isolation and manipulation
of cloned DNA fragments.
MEDICAL SCIENCE
Researchers use
electrophoresis to test variations
of vaccines with different levels
and types of antibodies to
conduct studies to find the best
possible version of a single
vaccine. Analysis of proteins and
antibodies: Another key
application for electrophoresis is
protein and antibodies analysis.
THANK YOU !!!!

DONE BY :
C.REJINA MPHARM 1ST SEM
DEPARTMENT OF PHARAMACOLOGY
KMCH COLLEGE OF PHARMACY