Electrophoresis

Electrophoresis is the migration of charged particles in an electric field

towards the electrode bearing the opposite charge.

 Electrophoresis is a method whereby charged molecules in solution,

chiefly proteins, and nucleic acids, migrate in response to an electrical
field.

 Biomolecules, like (DNA, RNA) nucleic acids, Amino acids, proteins, etc.
carry +ve /-ve charge with them.

 When these biomolecules are placed in an electric field then charged

molecules move towards the electrode of opposite charge due to the
phenomenon of electrostatic attraction.

 Molecules with +ve charge moves toward cathode & Molecules with -ve
charge move toward Anode.
 Principle
What is the principle of electrophoresis (1.5/20 + 17 + 15)
 Migration and separation of charged particles (ions) under the
influence of an electric field.

 An electrophoretic system consists of 2 electrodes of opposite

charge (anode, cathode), connected by a conducting medium
called an electrolyte.

 DNA is negatively charged when placed in an electrical field,

DNA will migrate toward the positive pole (anode).

DNA

• Polymerized agarose is porous,

allowing for the movement of DNA
 Agarose gel electrophoresis is a routinely used
method for separating proteins, DNA or RNA.

 The migration flow is determined solely by the

molecular weight where small weight
molecules are faster than larger ones.

 In order to visualize nucleic acid molecules in

agarose gels, ethidium bromide or SYBRGreen
are commonly used dyes.

 Illumination of the agarose gels with 300-nm

UV light issue was sequently used for
visualizing the stained nucleic acids.
 Strength of the electric field depends on
 the length of the gel
 potential difference at the ends (V/cm).

 The migration velocity is affected by

 the frictional force imposed by the gel matrix.
 Charge
 Size

3 factors influence the speed of movement

 the voltage of the electrical field,
 the concentration of agarose, and
 most importantly, the size of the DNA molecule

Charge to mass ratio is the same for DNA molecules of

different lengths hence, the size of the DNA, effectively
determines the rate at which it passes through the gel.
 Component
 The equipment required for electrophoresis consists basically of two items

 a power pack
 an electrophoresis unit

 Electrophoresis units are available for running either vertical or horizontal gel systems

 Vertical slab gel units are commercially available and routinely used to separate
proteins in acrylamide gels

 The gel is formed between 2 glass plates that are clamped together but held apart by
plastic spacers.

 The most commonly used units are the so-called mini gel apparatus

 Gel dimensions are typically 8.5 cm wide and 5 cm high, with a thickness of 0.51 mm

 A plastic comb is placed in the gel solution and is removed after polymerization to
provide loading wells for up to 10 samples.
 Factors Affecting Electrophoresis (2/18 + 20 +17)
1. Number of charges on molecule:
The velocity of molecule ∝ Number of charges
If an increasing number of charges then electrophoretic Velocity is also increased.
(Al^+3 > Mg^2+ > Na+)

2. Size & shape of the molecule:

Grater size -----------↓se mobility
Globular Shape -----------↑se mobility
Fibrous Shape ----------- ↓se mobility

3. Ionic strength of buffer:

↑ se ionic strength ↑se heat production ↓se mobility
↓se ionic strength less heat production ↑se mobility

4. pH of buffer solution:
pH ↓ → ion ↑ ∝ Current ↑
If pH is decreased then the concentration of ions will be increased & more current
will flow.
5. Temperature:[optimum temperature is 4°C]

By changing temperature, the following factors will

effects

•Viscosity of buffer gel

↑ temperature, ↓ viscosity

•Diffusion
↑ temperature, ↑ diffusion

•Evaporation
↑ temperature, ↑ evaporation

•Density
↑ temperature, ↓ density
The Net Charge is Determined by the pH of the Medium

• Proteins are amphoteric compounds, that is, they 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.

• Most of the charge of a protein comes from the pH-dependent

ionization of amino acid side-chain carboxyl and amino groups.

• Because these groups can be titrated over normal electrophoresis

pH ranges, the net charge of a protein is determined by the pH of
the surrounding medium and the number and types of amino acids
carrying amino or carboxyl groups.

• Nucleic acids, unlike proteins, are not amphoteric. They remain

negative at any pH used for electrophoresis.
 Supporting Media

Supporting media is a matrix in which the protein separation takes place

o Various type has been used for the separation either on a slab or capillary form

o Separation is based on the charge to mass ratio of protein separation

depending on the pore size of the medium, possibly molecular size.

 Aqueous protein solution is immobilized in solid hydrophilic support.

 Filter paper
 Cellulose acetate membrane
 Agar & agarose
 Starch
 Polyacrylamide

Nowadays electrophoretic technique is used in agarose gels and polyacrylamide

gels
 Major Types

Agarose gel electrophoresis Poly Acrylamide Gel Electrophoresis (PAGE)

(AGE)
 Agarose gel
Agarose is a linear polysaccharide made up of the basic repeat unit agarobiose,
which comprises alternating units of galactose and 3,6-anhydrogalactose.

It highly purified polysaccharide derived from agar (extracted from seaweed),

long sugar polymers held together by hydrogen and hydrophobic bonds.

Fig: Agarobiose, the repeating unit of agarose.

• The percentage of agarose included in a gel impacts the pore sizes and thus the size
of molecules that may pass through and the speed at which they do so.

• The higher the percentage of agarose, the smaller the pore size, thus the smaller the
molecules able to pass, and the slower the migration.

• In the molecular biology lab, 0.7-1% agarose gel is typically used for day-to-day DNA
separations, offering good, clear differentiation of fragments in the range of 0.2-10
kb.

Fig: Pore formation and temperature-induced state transition in agarose gel.

 Electrophoresis Apparatus

1. Electrophoresis is carried out in a tank suitable for

supporting medium e.g. paper or gel.
A major part of the tank include –
Buffer chamber
Electrodes
Electrical connection to the power supply
Space for keeping support medium
i.e. paper & slides of agar gel.
2. Power tank – to provide constant currents or constant
voltage.
 Electrophoresis Equipment

Power supply

Cover
Gel tank

Electrical leads

Casting tray
Comb
 The protocol can be divided into four stages

2. Sample and
1. Preparation Marking
of agarose gel Loading

3. Running The gel

Optimum voltage and 4. Staining the gel for
optimum time for viewing DNA
effective separation
 Making an agarose gel
An agarose gel is prepared by combining
agarose powder and a buffer (TAE or TBE)
solution.
Agarose gels are usually prepared using a Flask for boiling
weight/volume solution in the 0.5-2% range.
An optimal percentage of agarose gels will
result in the best separation and resolution of
bands (DNA fragments).

1. Weigh out the agarose and add it to the Agarose

flask/beaker containing the buffer.
2. For example, for a 1% agarose gel, add 1 g
agarose to 100 ml buffer.
3. Allow the agarose to sit in the solution for
a few minutes before swirling the
flask/beaker and suspending it in the
solution.
TAE buffer

TAE stands for Tris-acetate-EDTA. This buffer

contains Tris base, glacial acetic acid, and
EDTA. It is commonly used as a running buffer
in gel electrophoresis to separate nucleic acids.

When to Use TAE

 TAE produces a better separation of larger

fragments, which is greater than 3 kb.

 TAE works better for cloning because TBE

contains borate.

 Borate in TBE is an inhibitor for many

enzymes, such as ligase.

 TAE works better for performing DNA

extraction from agarose gel.
TBE Buffer

 TBE stands for Tris-borate-EDTA. It consists of Tris base,

boric acid, and EDTA.

 This buffer is commonly used in agarose gel

electrophoresis.

 In addition, it’s often used for analyzing DNA products

from PCRs by using agarose gel electrophoresis or
polyacrylamide gel electrophoresis.

When to use TBE

 TBE is suitable for obtaining a higher resolution of smaller

fragments, smaller than 2 kb. Using TBE for small
fragments provides sharper bands.

 TBE has a higher buffering capacity than TAE, so it works

better for a longer run.
 EDTA (ethylenediaminetetraacetic acid)
 EDTA (ethylenediaminetetraacetic
acid) is a chelating agent that binds
divalent metal ions such as calcium
and magnesium.

 In agarose gel electrophoresis, EDTA is

added to the buffer for chelating the
magnesium ions which are cofactors
for DNA nucleases.

 Hence, the activity of DNA nucleases

that may be present is inhibited, and
DNA is protected from degrading by
DNA nucleases.

Structure of (EDTA)
 Gel casting tray & combs Preparing the casting tray

 Seal the edges of the casting tray and put them in the combs. Place the
casting tray on a level surface.
 None of the gel combs should touch the surface of the casting tray.
 Agarose Buffer Solution

 Combine the agarose powder and buffer solution (1-­‐

2% agarose).
 Use a flask that is several times larger than the volume
of buffer.
 Swirl gently to give a uniform suspension(no lumps!).
 Melting the agarose

Agarose is insoluble at room The agarose solution is boiled until clear (right)
temperature left

• The easiest way to boil the mixture is with a microwave oven.

• Gently swirl the solution periodically when heating to allow all the
grains of agarose to dissolve.

• Be careful when boiling the agarose solution may become superheated

and may boil violently if it has been heated too long in a microwave
oven.
 Staining the Gel

• The most commonly used stain for visualizing

DNA is Ethidium bromide (EtBr)*.
• Alternative stains for DNA in agarose gels include
SYBR Gold, SYBR green, crystal violet, and
methyl blue.
• The sensitivities of methylene blue and crystal
violet are low compared with ethidium bromide.
• SYBR gold and SYBR green are highly sensitive
but more expensive than EtBr.

• Ethidium bromide is a powerful mutagen

and is moderately toxic.
• Gloves should be worn at all times.
• Dispose of the waste correctly
Ethidium bromide (EtBr)*.
 Pouring the gel

 Allow the agarose solution to cool  Each of the gel combs should be
by placing the flask in a water bath submerged in the melted
set at 55◦C. Add DNA dye, and mix  Agarose solution. Remove air
by swirling. bubbles with a pin or pipette tip.
 Then carefully pour the melted
agarose solution into the casting
tray. Avoid air bubbles.
  Place the gel in the
 When cooled, the agarose
electrophoresis chamber.
polymerizes, forming a flexible gel. It
should appear lighter in color when
completely cooled (30-­‐45
minutes).

 Carefully remove The combs and

tape.

 The gel can be used immediately or

stored (wrapped in cling film) in a
 Running buffer
 Running buffer is a conductive liquid that
allows the DNA to migrate through the
agarose gel. It is important that the agarose Direction of DNA migration
gel be made using the same buffer and
concentration. wells

Cathode Anode
Function of buffer (positive)
(negative)

 carries the applied current

 established the pH
 determine the electric charge on the solute
 High ionic strength of the buffer
 Produce the sharper band
 produce more heat

 Add enough electrophoresis buffer to cover the gel to

a depth of at least ~2 mm.
 Make sure each well is filled with buffer.
 Sample Preparation

Loading dye

Loading dye has 2 primary components:

 a visible dye (Bromophenol Blue)

indicates how far the DNA has run
on the gel Bromophenol Blue (for
color) Loading dye
 glycerol, which is denser than the
buffer, ensures that samples fall into
the loading
wells rather than float back out.
 Sample Preparation

 Mix the samples of DNA with 2X sample loading buffer containing a dye.
(Mix 1 part loading buffer with 1parts DNA sample).

 Approximately 5-­‐10 microliters (μL) of the sample are loaded into a well,
depending on the size of the well.

 The dye in the loading buffer allows the samples to be seen when loading
onto the gel. Glycerol in the loading buffer increases the density of the
samples, causing them to sink into the wells.

 The loading buffer dye, which migrates during electrophoresis, also helps
to monitor the progress of the separation.
 Loading the Gel

 Carefully place the pipette tip over a well and gently

expel the sample.
 The sample should sink into the well. Be careful not
to puncture the gel with the pipette tip.
 Running the Gel

o Place the cover on the electrophoresis chamber, connecting the electrical

leads.
o Connect the electrical leads to the power supply. Be sure the leads are
attached correctly

o DNA migrates toward the anode (red).

o When the power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
 DNA Ladders

 DNA Ladders, also known as molecular-

weight size markers, contain a set of
predetermined DNA fragment sizes.

 These markers are a set of standards used to

identify the approximate size and
concentration of a DNA fragment run on gel
electrophoresis.

 The most important use of 1 kb plus DNA

ladders is for the determination of the size of
double-stranded DNA in the range of 250 bp
to 25,000 bp.

 It helps to determine the size of DNA

fragments that are much longer than the Figure: 1 kb plus DNA ladder.
usual fragments.
DNA Ladders
 Electrophoresis Separates Dyes By Charge

 The sugar-phosphate backbone of DNA has

a strong negative charge.

 The current drives the DNA fragments

through the gel towards the positive
electrode.

 Small DNA fragments move through pores

easily, but large DNA fragments have a
more difficult time squeezing through the
tunnels.

 The DNA separates into discrete bands.

 The dyes separate into distinct zones based

on their charge.
 A Pattern of DNA under UV light

Ethidium bromide (EtBr)* SYBR green GelRed

Fig : Overview Of Gel Electrophoresis
 For 1% Agarose Gel:

 Take 1 gram of Agarose powder in a flask and mix with 100 ml of 1X TAE or TBE
buffer.

 Place the flask in Oven for 3 minutes at medium temperature

 Let the mixture cool down at room temperature

 Add 6 microliter Ethidium Bromide and pore gel to the casting tray

 Put Gel combs and let gel be hard

 Place gel in Gel tank

 Take 2 microliter DNA sample and mix with 2 microliter loading dye and load gel

 Run the gel for 30 minutes at 100 voltage

 Visualize gel in Gel dock

 APPLICATIONS
Write down the application of electrophoresis (1.5/15)
1. Separation of biological molecules like
plasma proteins, nucleic acids, nucleotides,
charged carbohydrate derivatives, glycoproteins,
lipoproteins, hemoglobin variants, isoenzymes
etc.

2. Analytical Applications-
 Determination of DNA sequences Analysis of
PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting.

 Separation of restriction digested genomic DNA

prior to Southern transfer, or of RNA prior to
Northern transfer.

 Southern & Northern Blotting

 Restriction Mapping of DNA
3. In Protein Study
 Determination of subunit stoichiometry
 Determination of molecular weight

4. Vaccine Analysis

 Vaccine analysis is one of the many important applications of

electrophoresis.

 There are several vaccines that have been purified, processed

and analyzed through electrophoresis, such as the influenza
vaccine, hepatitis vaccine and polio vaccine.
Advantage & Disadvantage of Agarose gel Electrophoresis

Advantage Disadvantage

 Nontoxic gel medium  Higher cost of agarose

 Gels are quick, easy  Fuzzy bands
 Poor separation of low
 Good for separating large molecular weight samples
DNA molecule
 Can recover samples by
melting the gel, digesting
with enzyme agarose or
treating with chaotropic
salts
 Polyacrylamide gels
 Acrylamide gels are used in polyacrylamide gels electrophoresis. These gels
are formed by the polymerisation of acrylamide monomer in the presence of
smaller amounts of N, N’-methylene-bisacrylamide (‘bis’-acrylamide) which
is used as a cross-linking agent.

 The polymerisation of acrylamide is an example of free-radical catalysis and is

initiated by the addition of ammonium persulphate and the base
tetramethylenediamine (TEMED). TEMED catalyses the decomposition of the
persulphate ion to give a free radical.

Fig: The formation of a polyacrylamide gel from acrylamide and bis-acrylamide.

 If this free radical is represented as R and M as an acrylamide monomer
molecule, then the polymerisation can be represented as follows:

R+ M → RM·
RM + M →RMM·
RMM + M → RMMM· etc.

 Long chains of acrylamide are built up, being cross-linked by the

introduction of the bis-acrylamide molecule into the growing chain. Then
the solutions are briefly placed under a vacuum to remove loosely
dissolved air prior to use.

 Photopolymerisation is an alternative method that can be used to

polymerise acrylamide gels.

 Riboflavin is used instead of ammonium persulphate and TEMED when

the gel is poured.

 Then it is placed in front of bright light for 23h.

 Photodecomposition of riboflavin generates a free radical that initiates
polymerisation.

 The pore size in the gel can be varied by changing the concentrations of both the
acrylamide and bis-acrylamide.

 Acrylamide gels can be made with a content of 3% - 30% acrylamide.

For example, Gels of 10% - 20% acrylamide are used in techniques such as SDS–
PAGE.

 The smaller pore size introduces a sieving effect that contributes to the
separation of proteins according to their size.
 The procedure of polyacrylamide gel electrophoresis

Preparation of polyacrylamide gel

Step 1:

 Gather combs, glass plates,

spacer (silicone tubing), and
binder clips.

 A comb is used to make wells

(lanes) to load samples.

 Use an appropriate comb

depending on the sample size.

Example: Use an 8-lane comb for 7

samples and molecular weight
markers.
Step 2:
 Thoroughly clean the glass plates with
ethanol, and assemble the gel casting mold.

Step 3:
 Pour acrylamide solution into a separating
gel.

 Overlay with water to prevent contact with

air (oxygen), which inhibits polymerization.

 Allow acrylamide to polymerize for 20-30

minutes to form a gel.

 Remove the overlaid water

Step 4:

 Polymerization of polyacrylamide gel. Proteins

migrate at different rate depending on the
concentration of the separating gel.

 Use an appropriate gel concentration for your target

protein.

 Using a higher acrylamide concentration produces a

gel with a smaller mesh size suitable for the
separation of small proteins.

 In general, an acrylamide concentration 6 - 15% is

used.

 Gels with an acrylamide concentration gradient

(gradient gels) are also used
Step 5 :

 Pour acrylamide solution into a stacking

gel; insert a comb and allow the
acrylamide to polymerize.

 Proteins are highly concentrated when

they migrate through a stacking gel prior
to entering a separating gel.

 The concentration occurs due to the

difference in the rate of migration of
glycine ion, chloride ion, and proteins.
 Preparation of samples

 Add sample buffer to samples,

and mix by flicking the tube.

 Heat the samples at 100°C for 3

minutes in a heat block.

 Centrifuge at 15,000 rpm for 1

minute at 4°C, and use the
supernatant for SDS-PAGE.
 Electrophoresis

 Remove the binder clips, spacer, and comb

from the gel assembly, and mount the gel in
the electrophoresis apparatus using binder
clips.

 Pour running buffer into the upper and lower

chambers of the electrophoresis apparatus,
and remove air bubbles and small pieces of
gel from the wells and under the gel using a
syringe.
 Load samples and molecular
weight markers in wells.

 Turn on the power supply, and run

the gel until the dye (BPB) in the
sample buffer reaches the bottom
of the gel.
 Remove the gel assembly
from the electrophoresis
apparatus.

 Remove the gel from the

glass plates using a spatula,
and prepare for subsequent
analysis.
 Applications

 Determine purity of protein samples

 Determine the molecular weight of the protein

 Identifying disulfide bonds between protein

 Quantifying proteins

 Blotting applications
 Advantages & Disadvantages of
Polyacrylamide gel electrophoresis

Advantage Disadvantage
 Toxic monomers
 Stable chemically cross-  Gels are tedious to prepare and often
linked gel leak
 Sharp bands  Need new gel for each experiment
 Good for separation of low
molecular weight fragments
 Comparison
Diiference between Agarose and PAGE (2/18)
 Agarose is poured horizontally, while
polyacrylamide is poured vertically.

 Agarose is more expensive than

polyacrylamide.

 Agarose consists of many molecules,

while polyacrylamide consists of only
one large molecule.

 Unlike polyacrylamide, agarose can be

re-heated.

 Agarose is considered a non-toxic

substance in all its forms, while the
powder of polyacrylamide is
determined to be toxic.
 Agarose mainly used to separate DNA and Polyacrylamide used to separate protein

 Agarose is used for large fragment ( more than 3 kbps ) and PAGE is used for small
fragment ( less than 2 kbps )
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE)
Mention the visualization process, application , advantage
and disadvantage (3/17 + 15)
o Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis
(SDS–PAGE) is the most widely used method for analyzing protein
mixtures qualitatively.

o It is particularly useful for monitoring protein purification and,

because the method is based on the separation of proteins according
to size, it can also be used to determine the relative molecular mass
of proteins.

o SDS is an anionic detergent and samples to be run on SDS–PAGE are

firstly boiled for 5 min in a sample buffer containing β-
mercaptoethanol and SDS.
 Principle
• SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, is a technique widely used in
biochemistry, forensics, genetics, and molecular biology to
separate proteins according to their electrophoretic
mobility.

• The SDS gel electrophoresis of samples having identical

charge to mass ratios results in fractionation by size and is
probably the world's most widely used biochemical
method

• Separates protein in an electric field.

• Migrate through a liquid or semisolid medium when

subjected to an electric field from anode to cathode
terminal

• Molecules flow at different rates depending on the

molecular size of proteins.
Fig: An apparatus used for SDS-PAGE
 Preparation of GEL
1. Clean the plates and combs.
2. Set up the plates on the rack.
3. Pour the separating gel ( Resolving
Gel ) .
4. Pour the stacking gel.
5. Gel storage.

Process of SDS-PAGE
4. Boil the samples for 10 minutes to
completely denatures the proteins.
5. Assemble the gel into the apparatus.
6. Pour the buffer solution into the
chamber ( Tris Glycerin Chloride ) .
7. Load 20uL of samples into the well.
8. After that, run electrophoresis by
connecting the current supplies.
 Visualization of protein bands

• Visualizes the band under UV light

• Types of Stains:

 Coomassie Blue
• Traditional method requires staining followed by de-staining to remove
background gel staining.
• Most common and least sensitive
• Limited to ~100ng of protein

 Silver Stain
• Most sensitive test
• Detection limit: 0.1-1.0ng of protein
A. Staining band with Western blot;
B. Coomassie blue stain;
C. Silver stain
 Application of SDS-PAGE:
Application of SDS-PAGE and advantage & disadvantage (2/20)
• It Is used for analyzing protein mixtures qualitatively, particularly useful for
monitoring protein purification and it can also be used to determine the relative
molecular mass of proteins.

• Used to measure the molecular weight of the molecules.

• used to estimate the size of the protein.

• Used in peptide mapping.

• used to compare the polypeptide composition of different structures

• Used to estimate the purity of the proteins. An obvious limitation of SDS-PAGE

resides in its deliberate denaturation of proteins prior to electrophoresis.

• Enzymatic activity, protein binding interactions, detection of protein cofactors,

etc. generally cannot be determined on proteins isolated by SDS-PAGE.
 Advantages & disadvantages of SDS-PAGE

Advantages Disadvantages

 Migration is proportional to  Poor band resolution due to

the molecular weight high alkaline operating pH
 Highly sensitive test,  Acrylamide gel is a potent
separates 2% difference in neurotoxin chemical
mass  Gel preparation is difficult
 Require a small number of  Requires a longer time
samples  Highly cost
 Stable chemically cross-
linked gel
Difference Between Gel Electrophoresis and SDS PAGE
Gel Electrophoresis SDS PAGE:

Composition Equal throughout the whole gel; made up of Two gels with different
agarose concentrations; made up of
polyacrylamide

Run Configuration Horizontal run Vertical run

Casting Methodology Sets as it cools Sets by a chemical reaction

Pore Size Pore size is not uniform; higher the agarose Pore size is uniform; the ratio of
concentration, smaller the pore size acrylamide to bis-acrylamide
determines the pore size

Concentration 0.5-2% 6-15%

Separation 50-20,000 bp nucleic acids 5-250,000 Da proteins

Staining With ethidium bromide Coomassie staining or silver staining

Conditions Generally run under native condition Denaturing conditions

Preparation Simple A complex process

 Native (buffer) gel electrophoresis:
 When one is aiming to detect a particular protein(often an enzyme) on the
basis of its biological activity, native (buffer) gel electrophoresis is most
frequently used.
 Because the protein(enzyme) is not denatured by the native (buffer) gel
electrophoresis procedure.
 Principle
 Almost similar to SDS-PAGE electrophoresis. In native or buffer gels,
polyacrylamide gels are again used (normally. a 7.5% gel) but the SDS is
absent and the proteins are not denatured prior to loading.
 Since all the proteins in the sample being analyzed carry their native charge
at the pH of the gel (normally pH8.7), proteins separate according to their
different electrophoretic mobilities and the sieving effects of the gel.

 Advantages
• The range of different charges and sizes of proteins in a given protein
mixture can be identified
• Biological activity of the protein of interest can be known Since the protein
is not denatured.
 Isoelectric Focusing
Principle of IEF (2/20)
 This method is also known as electro focusing, ideal for the separation of
amphoteric substances (Amphoteric compounds or ions that can react both
as acid and base) such as proteins.

 The electrophoretic method separates proteins according to the iso-electric

points.

 Each protein has its own isoelectric pH, pI = pH at which the protein has an
equal amount of positive and negative charges (the net charge is zero).

 Ideal for separation of amphoteric substances.

 Separation is achieved by applying a potential difference across a gel that

contains a pH gradient.

 Isoelectric focusing requires solid support such as agarose gel and

polyacrylamide gel.
 Isoelectric focusing gels contain synthetic buffers called ampholytes
that smooth the pH gradients.

 Ampholytes are complex mixtures of synthetic polyamino-

polycarboxylic acids.

 Commercially available ampholytes are-

 BIO-LYTE
 PHARMALYTE
 It gives good separation with a high resolution compared to any
other method.

 Resolution depends on

 The pH gradient,
 The thickness of the gel
 Time of electrophoresis,
 The applied voltage,
 Diffusion of the protein into the gel.

 Isoelectric focusing is the first step in two-dimensional gel

electrophoresis, in which proteins are first separated by their pI
and then further separated by molecular weight through SDS-
PAGE.
Advantages:

 The method has a high resolution, being able to separate proteins

that differ in their isoelectric points by as little as 0.01 of a pH unit.

 Isoelectric focusing is the first step in two-dimensional gel

electrophoresis
Two-dimensional polyacrylamide gel electrophoresis
Two-dimensional polyacrylamide gel electrophoresis
What do understand by 2D electrophoresis (2/18)

o This technique combines the technique of Isoelectric focusing (first

dimension), which separates proteins in a mixture according to charge (pI),
with the size separation technique of SDS–PAGE (second dimension).

o The combination of these two techniques to give a two-dimensional (2-D)

PAGE provides a highly sophisticated analytical method for analyzing
protein mixtures.

Fig: TWO DIMENSIONAL GEL ELECTROPHORESIS (2-DE)

2-DE separates proteins depending on two different steps
 The first one is called isoelectric focusing (IEF) which separates proteins
according to isoelectric points (pI);
 The second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which
separates proteins based on the molecular weights.

• Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for

proteomics work.
• It is used for the separation and fractionation of complex protein mixtures
from biological samples.

• Thus, thousands of proteins can be separated, and information about IEF and
molecular weights can be obtained. There are several steps for a successful 2-
DE analysis.
Mr = molecular weight
• For large-format gels, the first dimension (isoelectric focusing) is carried
out in an acrylamide gel that has been cast on a plastic strip (18 cm 3 mm
wide).

• The gel contains ampholytes (for forming the pH gradient) together with 8
M urea and a non-ionic detergent, both of which denature and maintain the
solubility of the proteins being analyzed. The denatured proteins therefore
separate in this gel according to their isoelectric points.

• The IEF strip is then incubated in a sample buffer containing SDS (thus
binding SDS to the denatured proteins) and then placed between the glass
plates of, and on top of, a previously prepared 10% SDS–PAGE gel.

• Electrophoresis is commenced and the SDS-bound proteins run into the gel
and separate according to size. The IEF gels are provided as dried strips and
need rehydrating overnight.

• The first dimension IEF run takes 68 h, the equilibration step with SDS
sample buffer takes about 15 min, and then the SDS–PAGE step takes about
5 h.
 Western Blotting
 Western Blotting is used to identify the large
molecular antigen (usually protein) that can
interact with specific antibodies and
determine the antigen of the MV (Measles
Virus) .

 The protein was separated by SDS

electrophoresis gel, and then transferred to
solid phase support by electrophoresis.

 Solid phase support includes nitrocellulose

membrane, poly vinylidene fluoride two
(PVDF) membrane, and cationic nylon
membrane.

 The unreacted sites on the membrane were

sealed up to inhibit the nonspecific
adsorption, so that the immobilized proteins
could interact with specific primary antibodies
and then the secondary antibodies which
conjugated with enzyme or fluorescein.
 Finally, it is located by means of radiation,
chromophore, or chemiluminescence.
 Protein (western) blotting

Fig. The use of enzyme-linked second antibodies in immunodetection of

protein blots.

First, the primary antibody (e.g. raised in a rabbit) detects the protein of
interest on the blot.

Second, enzyme-linked anti - rabbit IgG detects the primary antibody.

Third, the addition of enzyme-substrate results in colored

product deposited at the site of protein of interest on the blot.
Write down the principle of capillary electrophoresis (1/17)