Electrophoresis

Mr. R. K. Lodha
Assistant Professor,
Department of Biotechnology,
PVP College, Pravaranagar
Mr. R. K. Lodha

KEY CONCEPTS
•General principle
•Factors affecting Electrophoresis
•Types of Electrophoresis
•Gel Electrophoresis
•Applications
Mr. R. K. Lodha

Why electrophoresis?
–To determine the number,
amount and mobility of
components in a given sample
or to separate them.
–To obtain information about
the electrical double layers
surrounding the particles.
–Determination of molecular
weight of proteins and DNA
sequencing
Mr. R. K. Lodha

This electrokinetic phenomenon was observed for the first time in
1807 by Ferdinand Frederic Reuss (Moscow State University), who
noticed that the application of a constant electric field caused clay
particles dispersed in water to migrate.
History of Electrophoresis
Mr. R. K. Lodha

INTRODUCTION
ELECTROPHORESIS― Electro +
Electric Field
Phoresis
Migration
Electrophoresis is used in molecular biochemistry,
microbiology, biomedical research.
It is a type of separation method.
It is one of the highly efficient technique of analysis
and method for separation of biomolecules.
It is both qualitative and quantitative analysis
technique.
It is similar to chromatography but differs in types of
samples used, principle used etc.
Mr. R. K. Lodha

•Electrophoresis: Differential movement or migration of
charged molecules (ions) in solution, with response to
an electrical current.
•Separation of molecules according to size and/or
charge.
•Negatively charged molecules (anions) will be attracted
towards anode.
•Positively charged molecules (cations) will move
towards cathode.
GENERAL PRINCIPLE
Anode
Cathode
Mr. R. K. Lodha

•As an analytical tool,
electrophoresis is simple, rapid
and highly sensitive.
•Rate of migration depends on:
 Molecular charge (net charge)
Molecular shape and size
Strength of the electrical field,
Ionic strength, viscosity, and
temperature of the medium.
BASIC CONCEPT
https://upload.wikimedia.org/wikipedia/commons/
a/ab/Electrophoresis.svg
Mr. R. K. Lodha

• The force (F) experienced by a particle in an electrical
field is given by Coulomb’s law,
F = Ze E
(Where, E - Electric field: potential per unit length)
• The viscous resistance of the medium to the motion: -fv
(Where, f - Frictional factor)
• The viscous resistance of the medium just balances the
driving force.
-fv = F = Ze E
GENERAL PRINCIPLE
• An isolated charged particle in a non-conducting medium.
Mr. R. K. Lodha

On the application of electric field, the
suspended particles exerts an electrostatic
coulomb force(FEle) which is given by―
FEle=q × E
A drag/retardation force(FRet) will also be
immediately exerted on the particles by the
medium whose direction is opposite to that of
particle movement is given by―
FRet=f × v
When electrophoresis is started, particles accelerate instantaneously to a
velocity(v) at which FEle & FRet become equal
i.e. FEle = FRet q × E = f × v v/E = q/f
The velocity of a particle when one unit of electric field is applied is called
the Electrophoretic mobility(μe) of that particle and given as― μe = v/E
ELECTROPHORETIC THEORY
Mr. R. K. Lodha

• The rate of migration of the molecule
v = Eq/f
Where, v = molecule velocity
E = Electric field strength
q = molecular charge
f = friction coefficient of molecule
ELECTROPHORETIC THEORY
• Electrophoretic mobility value (µ):
µ = v/E = Eq/Ef = q/f
• From above equation, molecules move through gel based on
charge to friction ratio
– Since friction is based primarily upon mass, molecules
migrate based upon charge to mass ratio
– Therefore, differences in µ approximate differences in mass.
Mr. R. K. Lodha

• Electric current is carried by buffers
– Buffers keep the pH and charge surrounding
analyte constant.
• Effects of the electric field on the sample:
– In electrophoresis either current, voltage, or
power, is always held constant.
– Higher voltage causes greater migration speed.
– Also leads to generation of heat.
• May denature protein sample and destroy gel matrix
• Also mixes samples through convection of buffer
ELECTRIC FIELD
Mr. R. K. Lodha

Interrelation of Resistance, Voltage, Current
and Power
• Two basic electrical equations are important in electrophoresis
 The first is Ohm's Law, I = E/R
 The second is P = EI
 This can also be expressed as P = I2R
• In electrophoresis, one electrical parameter, either current, voltage, or
power, is always held constant
Mr. R. K. Lodha

• Under constant current conditions (velocity is
directly proportional to current), the velocity of the
molecules is maintained, but heat is generated.
• Under constant voltage conditions, the velocity
slows, but no additional heat is generated during
the course of the run.
• Under constant power conditions, the velocity
slows but heating is kept constant.
Mr. R. K. Lodha

 Performs multiple functions:-
 Carry current and prevents analyte from being
altered.
 Maintains the pH.
 Determine the electrical charge on solute
 Example- Common buffers are Tris-HCl and Tris-glycine.
– Tris-Borate-EDTA(TBE), Tris-Acetate-EDTA(TAE), Tris-
Phosphate-EDTA(TPE) used most often for DNA.
– 10 mM sodium phosphate buffer used for RNA.
 Buffer additives modify sample molecules.
– Formamide, urea (denaturing agents)
ELECTROPHORESIS BUFFERS
Mr. R. K. Lodha

• The net charge of the molecule is determined by the
pH of the medium.
• Proteins are amphoteric in nature (contain both acidic
and basic residues).
• Each protein has its own characteristic charge
properties depending on the number and kinds of
amino acids carrying amino or carboxyl groups.
• Nucleic acids, unlike proteins, are not amphoteric.
They remain negative at any given pH.
NET CHARGE AND PH
Mr. R. K. Lodha

Temperature and Electrophoresis
• Important at every stage of electrophoresis
• During Polymerization
 Exothermic Reaction
 Gel irregularities
 Pore size
• During Electrophoresis
 Denaturation of proteins
 Smile effect
 Temperature Regulation of Buffers
Mr. R. K. Lodha

It inhibits convection and diffusion, which would
otherwise impede separation of molecules.
It allows a permanent record of results through
staining after run.
It can provide additional separation through
molecular sieving.
What is the Role of the Solid Support Matrix?
Mr. R. K. Lodha

External Factors
• Voltage & Temperature
in voltage & temperature
= speed = heat and
leads to Protein denaturation
• Buffer pH
pH determines net charge of the
protein, hence direction of migration.
• Supporting medium
- Protein interaction slows speed
10
Internal Factors
• Charge of the molecule
in charge = “faster speed”
• Size and Shape
in size = “slower speed"
FACTORS AFFECTING ELECTROPHORESIS
The speed and direction of a moving charged particle influenced by
Mr. R. K. Lodha

TYPES OF ELECTROPHORESIS
Mr. R. K. Lodha

11
Electrophoresis
Zone Electrophoresis
• Paper Electrophoresis
• Capillary Gel Electrophoresis
• Gel Electrophoresis
- Agarose gel (DNA & Protein)
- Polyacrylamide gel (PAGE)
- SDS-PAGE (Protein)
Moving Boundary Electrophoresis
• Capillary Electrophoresis (CE)
Used to separate:
Proteins
Peptides & Amino acids
Inorganic ions
Organic bases & acids
Whole cells
Nucleic acids
TYPES OF ELECTROPHORESIS
Mr. R. K. Lodha

Gel Electrophoresis
It is a technique used for the separation of DNA, RNA or
protein molecules according to their size and electrical
charge using an electric current applied to a gel matrix.
What is a gel?
Gel is a cross linked polymer whose composition and
porosity is chosen based on the specific weight and
porosity of the target molecules.
Types of Gels:
Agarose gel.
Polyacrylamide gel.
Starch gel Mr. R. K. Lodha

TYPES OF GEL:
AGAROSE
• Used for the separation of proteins that are larger than 200 kDa.
• Samples are also easily recovered.
POLYACRYLAMIDE
• Polyacrylamide gel electrophoresis (PAGE) is used for separating
proteins ranging in size from 5 to 2,000 kDa
• Care must be used when creating this type of gel, as acrylamide is
a potent neurotoxin in its liquid and powdered forms.
STARCH
• The gels are slightly more opaque than acrylamide or agarose.
• They are visualised using Napthal Black or Amido Black staining.

Charge
Separation
Size
Separation Identify
Purify
Mixture of
Charged
Molecules
Negative
Molecules
Separation of a mixture of charged molecules
• A thin layer or zone of the macromolecule solution is electrophoresed
through solid support matrix (Gel).
• Charged molecules are separated based on their charge and size.
Positive
Molecules
Analyze
GEL ELECTROPHORESIS
https://slideplayer.com/slide/8432364/26/images/4/Separation+of+a+Mixture+of+Charged+Molecules.jpg
Mr. R. K. Lodha

GEL ELECTROPHORESIS
Separation based on the Size:
• The porous gel matrix act as a sieve to separate the molecules.
• By adjusting the pH of the buffer, molecules being separated
carry a net negative charge and will move towards the anode.
• As they move through the pores of the gel, the smaller
molecules move faster than the larger molecules.
SEM photoof 1% Agarose
(gel Matrix)
http://stevegallik.org/cellbiologyolm_gelelectrophoresis.html
http://www.bioscience-beads.com/underivatized.html
Mr. R. K. Lodha

• Can be poured into slabs and columns, can be drawn
into capillaries.
• Very stable, allowing for post-separation manipulation.
• Pore size can also be controlled for, altering the
migration properties of the gel.
• Two forms of gel matrices are used, cross-linked and
non-crosslinked.
• Most common cross-linked gels are agarose and
acylamide
– Agarose is a reversible matrix cross-linked by hydrogen
bonds
– Acrylamide is a permanent matrix cross-linked with
methylene bridges
THE PROPERTY OF GELS
Mr. R. K. Lodha

GEL ELECTROPHORESIS
Gel Electrophoresis: Supporting medium is GEL
Gels are composed of polymers of sugars (Agarose or Polyacrylamide)
• Agarose – a complex sugar chain from red seaweed.
• It has a large pore size good for separating large molecules.
• Polyacrylamide – chain of acrylamide molecules.
• It has a small pore size good for separating small molecules.
• 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.
• Electrophoresis of biological macromolecules is at present
carried out on either polyacrylamide or agarose gels
Mr. R. K. Lodha

Agarose Gel
• Separates fragments based on
mass and charge.
• They have large pore sizes and
are used for separating larger
DNA molecules (RFLP
Analysis) or RNAseparation.
• Typically resolve 200 bp-20 kbp •
• Also used to separate large
proteins and protein
complexes.
ELECTROPHORESIS OF DNA
Polyacrylamide (PAGE)
• Used to obtain high resolution
separations.
• Used for the separation of
smaller DNA molecules (STR
analysis and DNA sequence
analysis.
They have small pore size
gel, is used to separate most
proteins and small
nucleotides.
• Separates fragments < 200 bp.
Mr. R. K. Lodha

Agarose and Polyacrylamide
 Although agarose and polyacrylamide differ greatly in their
physical and chemical structures, they both make porous
gels.
 A porous gel acts as a sieve by retarding or, in some cases,
by completely obstructing the movement of
macromolecules while allowing smaller molecules to
migrate freely.
 By preparing a gel with a restrictive pore size, the operator
can take advantage of molecular size differences among
proteins
 Both are relatively electrically neutral
Mr. R. K. Lodha

What isAgarose?
 Alinear carbohydrate polymer extractedfrom
seaweed, agarobiose
 forms aporous matrix asit gels
– shifts from random coil in solution to structure in
which chainsare bundled into doublehelices
Mr. R. K. Lodha

 A highly purified uncharged polysaccharide derived from
agar.
 It is a linear polymer made up of the repeating unit of
agarobiose, which is a disaccharide made up of alternating D-
galactose and 3,6-anhydro-α-L-galactopyranose linked by α-
(1→3) and β-(1→4) glycosidic bonds.
Structure of the Repeating Unit of Agarose
Mr. R. K. Lodha

Agarose Gels
 Agarose is a highly purified uncharged polysaccharide
derived from agar
 Agarose dissolves when added to boiling liquid. It remains
in a liquid state until the temperature is lowered to about
40° C at which it gels.
 The pore size may be predetermined by adjusting the
concentration of agarose in the gel.
 Agarose gels are fragile, however they are actually
hydrocolloids and are held together by the formation of
weak hydrogen and hydrophobic bonds.
Mr. R. K. Lodha

Structure of the Repeating Unit of Agarose
G: 1,3-β-d-galactose A: 1,4-α-l-3,6-anhydrogalactose
Basic disaccharide repeating units of agarose
Mr. R. K. Lodha

TYPES OF AGAROSE
• Standard Agarose - LE
 Gels at 35-38oC; Melts at 90-95oC
 Becomes opaque at high concentrations
• Low Melting Agarose (NuSieve)
 Gels at 35oC; Melts at 65oC
 Often used to isolate DNA fragments from gel
Intermediate forms or combinations of LE and NuSieve
can provide sturdy, translucent gels at high agarose
concentrations .
Mr. R. K. Lodha

Concentrations of agarose used
% Agarose (w/v) Size range (kb pairs) for
optimal separation
2-30
0.7-20
0.5-10
0.2-3
0.1-2
0.07-1.5
0.04-0.9
0.03-0.6
0.01-0.4
•
•
•
•
•
•
•
•
•
0.5
0.75
1.0
1.5
2.0
3.0(Nu-Sieve)
4.0(N-S)
5.0(N-S)
6.0(N-S)
Low conc. = larger pores better resolution of larger DNA fragments
High conc. = smaller pores better resolution of smaller DNA
fragments
Mr. R. K. Lodha

Advantages:
Easy to prepare and small concentration of agar is required.
Resolution is superior to that of filter paper.
Large quantities of proteins can be separated and recovered.
Adsorption of negatively charged protein molecule is negligible.
It adsorbs proteins relatively less when compared to other medium.
Sharp zones are obtained due to less adsorption.
Recovery of protein is good, good method for preparative purpose.
Disadvantages:
Electro osmosis is high.
Resolution is less compared to polyacrylamide gels.
Different sources and batches of agar tend to give different results
and purification is often necessary.
Mr. R. K. Lodha

GEL ELECTROPHORESIS APPARATUS AND TYPES
• Horizontal Gel Units (“Submarine Gels”)
– Agarose gels
– Most DNA and RNA gels
• Vertical Gel Units
– Polyacrylamide gels (PAGE)
– Typically sequencing gels
• Pulse Field Gel Units
– Any electrophoresis process that uses more than one
alternating electric field
– Agarose
– Large genomic DNA (Chromosomal)
Mr. R. K. Lodha

AGAROSE GEL
ELECTROPHORESIS
Mr. R. K. Lodha

+
-
Power
DNA
H O

2

• DNA is negatively charged.
•When placed in an electrical field, DNA will migrate toward the
positive pole (anode).
• An agarose gel is used to slow the movement of DNA and separate
by size.

+
-
Power
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA
small
large

Components of an Electrophoresis System
 Power supply and chamber, a source of negatively charged
particles with a cathode and anode
 Buffer, a fluid mixture of water and ions
 Agarose gel, a porous material that DNA migrates through
 Gel casting materials & Comb
 DNA ladder, mixture of DNA fragments of known lengths
 Loading dye, contains a dense material and allows
visualization of DNA migration
 Staining agent (dye) DNA Stain, allows visualizations of
DNA fragments after electrophoresis
 Sample to be separate

*
Casting tray
Gel combs
Gel tank Cover
Electrical leads

Gel Electrophoresis Materials:
Hardware
Power supply
Mr. R. K. Lodha

GelCastingTrays
 Available in a variety of
sizes and composed of
UV-transparent plastic.
 The open ends of the
trays are closed with
tape while the gel is
being cast, then
removed prior to
electrophoresis.
Mr. R. K. Lodha

Appliedvoltage
  voltage,  rate of migration
 The higher the voltage, the more quickly the
gel runs
 But if voltage is too high, gel melts
 The best separation will apply voltage at no
more than 5V/cm of gel length.
Mr. R. K. Lodha

• During electrophoresis water undergoes hydrolysis :
H2O H + OH-
• Buffers prevent the pH from changing by reacting with the
H+ or OH- products
• Most common buffer used is called TRIS –
[tris(hydroxymethyl)aminomethane]
• Compound added to make Tris an effective buffer is— either
boric or acetic acid
• Compound added to bind metals is- EDTA
• The buffer is either TBE or TAE
o TBE is made with Tris/Boric Acid/EDTA
o TAE is made with Tris/Acetic Acid/ EDTA
Mr. R. K. Lodha

 TAE (Tris-acetate-EDTA) and TBE (Tris-borate-
EDTA) are the most common buffers for duplex DNA
 Establish pH and provide ions to support conductivity
 Concentration affects DNA migration
 Use of water will produce no migraton
 High buffer conc. could melt the agarose gel
 New Sodium Borate (SB) buffer allows gels to be run
at higher voltages in less time than traditional buffers
Electrophoresis Buffer
Mr. R. K. Lodha

AComb
 A comb is placed in
the liquid agarose
after it has been
poured
 Removing the comb
from the hardened
gel produces a
series of wells used
to load the DNA
Mr. R. K. Lodha

DNALadder
 It is a solution of DNA molecules of different
length.
 Since agarose gels separate DNA according to
size, the Mr of a DNA fragment may be
determined from its electrophoretic mobility by
running a number of standard DNA markers of
known Mr on the same gel.
 DNA Ladder consists of known DNA
sizes(sample of bacteriophage λ DNA (49 kb) that
has been cleaved with a restriction enzyme such
as EcoRI) used to determine the size of an
unknown DNA sample.
 Since the base sequence of λ DNA is known, and
the cleavage sites for EcoRI are known, this
generates fragments of accurately known size
which when run on an agarose gel looks like a
"ladder".
Mr. R. K. Lodha

 Contains a dense substance, such as
glycerol, to allow the sample to "fall"
into the sample wells.
 Contains one or two tracking dyes,
which migrate in the gel and allow
monitoring of how far the
electrophoresis has proceeded.
DNA samples are loaded into a gel AFTER the tank
has been filled with buffer, covering the gel.
Loading Dye
Mr. R. K. Lodha

AGAROSE GEL ELECTROPHORESIS-METHOD
Preparation of Agarose gel
Melt, cool and add Ethidium bromide. Mix thoroughly.
Pore into casting tray with comb and allow it to solidify
Add running buffer. Load samples and DNA markers
Run gel at constant voltage until band separation occurs
Observe the separated DNA bands in a UV chamber
Mr. R. K. Lodha

Agarose Buffer Solution
 Combine the agarose powder and buffer solution.
 Use a flask that is several times larger than the volume of
buffer.
STEPS
Mr. R. K. Lodha

Agarose, dissolved in gel buffer by boiling.
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Melting the Agarose
Mr. R. K. Lodha

Casting of the gel
 Agarose, dissolved in gel buffer by boiling, is poured onto a
glass or plastic plate, surrounded by a wall of adhesive tape or a
plastic frame to provide a gel about 3 mm in depth.
 Loading wells are formed by placing a plastic well-forming
template or comb in the poured gel solution, and removing this
comb once the gel has set.
Mr. R. K. Lodha

Sample Preparation
• Samples are prepared by dissolving
them in a buffer solution that
contains sucrose, glycerol or Ficoll,
which-
 makes the solution dense
 allows it to sink to the bottom of the
well.
• A dye such as bromophenol blue is
also included in the sample solvent;
 it makes it easier to see the sample
that is being loaded
 acts as a marker of the
electrophoresis front.
Mr. R. K. Lodha

Loading the Gel
The gel is placed in the electrophoresis tank, covered with
buffer, and samples loaded by directly injecting the sample into
the wells.
Mr. R. K. Lodha

*Dye DNA and place into gel
The gel is made out of
agarose, which is similar
to jelly.
The gel is made with
wells at one end so that
the DNA can be loaded
into the gel.
Mr. R. K. Lodha

buffer 
Cathode
(negative)
Anode
(positive)

wells
  
DNA
SETUP
Mr. R. K. Lodha

Running the Gel
General purpose gels are approximately 25 cm long and 12 cm wide,
and are run at a voltage gradient of about 1.5 V/ cm overnight.
Mr. R. K. Lodha

Staining of DNA
 Allows DNA visualization after gel
electrophoresis
 The favorite—Ethidium bromide
 When bound to DNA it fluoresces under
ultraviolet light (reddish –orange colour)
 Convenient because it can be added directly to
the gel
 Sensitive—detects 0.1ug of DNA
Mr. R. K. Lodha

Ethidium bromide
• Ethidium bromide is a cyclic
planar fluorescent dye that
intercalates between bases of
nucleic acids and allows very
convenient detection of DNA
fragments in gels.
• Inserting itself between the base
pairs in the double helix
• The standard concentration used
in staining DNA in gels is 0.5-
1ug/mL
Mr. R. K. Lodha

 UV absorbance maxima at 300 and 360 nm and
emission maxima at 590 nm.
 Detection limit of bound DNA is 0.5-5 ng/band.
 Ethidium bromide is mutagenic so care must be
taken while handling the dye.
 Othe alternatives for ethidium bromide :
 Methylene blue
 Syber safe
 Xylene cyanol
 Bromphenol blue
Ethidium bromide
Mr. R. K. Lodha

After electrophoresis the gel is
illuminated with an ultraviolet
lamp to view the DNA bands.
The ethidium bromide
fluoresces reddish-orange in
the presence of DNA.
As little as 10 ng of DNA can
be visualised as a 1 cm wide
band.
Photograph it with a digital
camera/ Gel-Doc.
Visualization
Mr. R. K. Lodha

 Smaller pieces of DNA travel farther than Larger
pieces of DNA.
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
Analysis
Mr. R. K. Lodha

Complete Process
https://en.wikipedia.org/wiki/Gel_electrophoresis_of_nucleic_acids
https://www.researchgate.net/figure/Preparation-loading-
and-running-of-gel-in-electrophoresis_fig1_224829868
The Gel with UV illumination
Image of the Gel
Mr. R. K. Lodha

Sample
Charge : Rate of migration increases with increase in net charge.
It depends on pH.
Size : Rate of migration decreases for longer molecules. It is due
to increase frictional and electrostatics forces.
Shape : Molecular have similar charge but differ in shape
exhibits different migration rate.
Globular substances move faster than the fibrous ones.
Mr. R. K. Lodha

Separation of Deoxyribonucleic acid
Separation of ribonucleic acid
Separation of protein molecules
It may be used as preparative technique prior to use of other
methods such as mass spectroscopy, cloning, DNA Sequences,
Southern Blotting for further characterization.
Separation of amino acid
Separation of lipoproteins
Separation of enzyme in blood
Separation of antibiotic drug
Used for estimation of molecular weight of proteins and nucleic
acids.
Determination of subunit structure of proteins.
Monitoring changes of protein content in body fluids.
Mr. R. K. Lodha

• Used to study the properties of a single charged species
or mixtures of molecules.
• Used to separate organic bases, acids and inorganic ions.
• Used to identify amino acids, peptides and proteins.
• Used to separate very large proteins, nucleic acids and
nucleoproteins etc.
• Used in Clinical Laboratory to separate proteins from
each other
– Proteins analysis in body fluids: Serum, Urine, CSF
– Proteins in erythrocytes: Hemoglobin
– Nucleic acids: DNA, RNA
ELECTROPHORESIS - APPLICATIONS
Mr. R. K. Lodha

• Agarose Gel electrophoresis is used to visualize:
– Genomic DNA
– RNA
– PCR products
– Plasmids
– Restriction enzyme digest products
ELECTROPHORESIS- APPLICATIONS
Mr. R. K. Lodha

References
Instrumental methods of chemical analysis. By Dr.
B.K.Sharma, Page no. 661-670.
Instrumental analysis by William Kemp.
Principle & techniques in biochemistry & molecular biology-
Wilson & Walker
http://www.intechopen.com/books/gelelectrophoresisprinciples
-and-basics
Gel electrophoresis & its applications by Pulimamidi rabindra
reddy and Nomula Raju.
Mr. R. K. Lodha

Electrophoresis

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