Dr.

D Y Patil Arts Commerce And Science College

Pimpri Pune 411018

Chemistry Department

Mrs. Tarannum S Attar

 CONTENT
Introduction
Principle
Factors affecting
Conventionalelectrophoresis
General operation
Technical and practicalConsideration
Types ofelectrophoresis

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 INTRODUCTION
Electrophoresis is the migration of charged particles
or molecules in a medium under the influence of an
applied electric field.

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The rate of migration of an ion in electrical field depend on
factors,
o Net charge of molecule
o Size and shape of particle
o Strength of electrical field
o Properties of supporting medium
o Temperature of operation
o Ionic strength of buffer
 Effect of heat

Peak broadening of samples

Decrease resolution

Formation of temperature gradient –convection current -

resulting mixing of analytes

Denaturation of enzymes and proteins

H=I2R
Electro endosmosis
Anode Cathode
 Conventional electrophoresis

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 Buffer
The buffer in electrophoresis hastwofold purpose:
Carry applied electricalcurrent
Theyset the pH atwhich electrophoresis is carried out.
Thus they determine;
Type of charge on solute.
Extent of ionizationof solute
Electrode towards which the solute will migrate.
The buffer ionic strength will determine the thickness of the
ionic cloud.
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 Commonly buffersused;
Buffer pH value

Phosphate buffer around 7.0

Tris-Borate-EDTA buffer(TBE) around 8.0

Tris-Acetate EDTAbuffer(TAE) above 8.0

Tris Glycine buffer(TG) more than 8.5

Tris -Citrate-EDTAbuffer (TCE) around 7.0

Tris -EDTAbuffer (TE) around 8.0

Tris -Maleic acid -EDTAbuffer (TME) around 7.5

Lithium Borate - buffer (LB) around 8.6

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 Supporting medium
Supporting medium is a matrix in which the
protein separation takes place.

Various type has been used for the separation

either on slab or capillary form.

Separation is based on charge and mass of

protein,depending on the pore size of the
medium.
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Chemical nature Inert

Availability easy
Electrical conductivity high
Adsorptivity low
Sievingeffect desirable
Porosity controlled
Transparency high

Electro-endosmosis(EEO) low

Preservation feasible
Toxicity low

Preparation easy
 - Starch gel
- Celluloseacetate
- Agarose
- Polyacrylamide gel

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 TYPES OF ELECTROPHORESIS
1) Zone Electrophoresis
a)Paper Electrophoresis
b)Gel Electrophoresis
c)Cellulose acetate Electrophoresis
d)Isoelectric Focusing
e) ImmunoElectrophoresis
2) Moving Boundary Electrophoresis
a)Capillary Electrophoresis
b)Isotachophoresis
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PAPER ELECTROPHORESIS
 Filter paper such as Whatmann no1 and no 3mm in strip of 3 or 5cm
wide have been used.

 Separation takes place in 12 to 14hrs.

ADVANTAGES:
 It is economical.

 Easy to use.

DISADVANTAGES:
 Certain compounds such as proteins, hydrophilic molecules cannot be
resolved properly due to the adsorptive and ionogenic properties of paper
which results in tailing and distortion of componentbands.

 Electro osmosis.
 Gel electrophoresis

 Separation is brought about through molecular sieving technique,

based on the molecular size of the substances. Gel material acts as
a "molecular sieve”.

 Gel is a colloid in a solid form (99% is water).

 It is important that the support media is electrically neutral.

 Differenttypes of gels which can be used are; Agar and Agarose

gel, Starch, Polyacrylamide gels.
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 Agarose Gel
 A linear polysaccharide (made-up of repeat unit of
agarobiose-alternating unit of galactose and 3,6-
anhydrogalactose).

 Used in conc as 1% and 3%.

 The gelling property are attributed to both inter- and
intramolecular hydrogen bonding

 Pore size is controlled by the % of agarose used.

 Large pore size are formed with lower conc and vice versa.

 Purity of the agarose is based on the number of sulphate

conc, lower the conc of sulphate higher is the purity of
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agarose.
 ADVANTAGES: DISADVANTAGES:
Easy to prepare and small
concentration of agar is required.  Resolution is less compared
to polyacrylamide gels.
Resolution is superior to that of
filter paper.  Different sources and batches of

Large quantities of proteins can be agar tend to give different

separated and recovered. results and purification is often

It adsorbs proteins relatively less necessary.

when compared to other medium.

Sharp zones are obtained due to less

adsorption.

APPLICATION:
To separate DNA, proteins, Hb variants, iso-enzymes etc.

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4
 TYPES OF ELECTROPHORESIS
1) Zone Electrophoresis
a)Paper Electrophoresis
b)Gel Electrophoresis
c)Cellulose acetate Electrophoresis
d)Isoelectric Focusing
e) ImmunoElectrophoresis
2) Moving Boundary Electrophoresis
a)Capillary Electrophoresis
b)Isotachophoresis
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 POLYACRYLAMIDE GEL ELECTROPHORESIS

Frequently referred to as PAGE.

Cross-linked polyacrylamide gel are formed from the
polymerization of the monomer in presence of small amount
of N,N”-methylene- bisacrylamide.

Bisacrylamide – two acrylamide linked by the methylene group.

The polymerization of the acrylamide is an example for free
radical catalysis.
Made in conc. between 3-30% acrylamide.
low % has large pore size and vice versa.

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 SDS-PAGE
Sodium dodecyl sulphate- polyacrylamide
gel electrophoresis.
Most widely used method for analysing protein
mixture qualitatively.
Useful for monitoring protein purification – as separation
of protein is based on the size of the particle.
Can also be used for determining the relative molecular
mass of a protein.

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 The sample is first
boiled for 5min in
SDSisananionic buffercontaining
detergent. • Beta-
Mercaptoethanol
• SDS

Mercaptoethanolwill break the disulphide bridges.

SDSbindsstronglyto anddenatures the protein.

Each protein is fully denatured and open into rod-shape with

series of negativelychargedSDSmoleculeon polypeptide chain.
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 On average, One SDS molecule bind for every two
amino acid residue.
Hence original native charge is completely swamped by
the negative charge of SDS molecule.

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 Components

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Stacking gel:

ordering/arranging and conc. the macromolecule before

entering the fieldof separation. (4% ofacrylamide)
Purpose is to concentrate protein sample in sharp band
before it enters main separating gel.

Running gel:

the actual zone of separation of the particle/molecules based

on their mobility. (15%of acrylamide)
 Movement ofparticle

[Cl] > [protein-SDS] > [Glycinate]

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APPLICATION:
- Research tool
- Measuring molecular weight
- Peptide mapping
- Protein identification
- Determination of samplepurity
- Separation ofproteins and
establishing size
- Blotting
- Smaller fragments ofDNA
ADVANTAGES:
-Gels are stable over wide range of pH and
temperature.
-Gels of different pore size can be formed.
-Simple and separation speed is good comparatively.
-Exhibit reasonablemechanicalstrength
-Low endosmosiseffect

DISADVANTAGES
-Gel preparation and casting is time-consuming
-carcinogenic
-Complete reproducibility of gelpreparation not possible
STAINING:
Fluorescent stains - Ethidium bromide – Nucleic acids
Silverstain for protein gel(sensitive50 times dyebased)
Dyebased– Coomassieblue–protein band
Trackingdyes– BPB>xylenecyanol
 Native (buffer)gel
Done byusingthe polyacrylamidegel(7.5%).
SDS is absent.

pH of8.7
Proteins are separated accordingto the electrophoretic
mobility &Sievingeffect of the gel.

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 Isoelectric focussing
First described by- H.Svensson in Sweden.
Method is ideal for the separation of the
amphoteric substances.

Method has high resolution.

Able to separate the protein which differ in isoelectric
point by as little as little 0.01 of pH unit.

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 Different gradient of thepH along
the length of the separatinggel.

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 Establishment of phgradient:

This is achieved by the ampholyte & must have following

prop:
Must dictate pH course (buffering capacity at their pI)
Should have conductance at their pI.

Low molecular weight

Soluble in water
Low light absorbance at 280nm.

Available commercially with pH band (3-11)

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Eg: Ampholine, Pharmalyte and Bio-lyte.
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 Movement

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 Application:
- Highlysensitivefor studyingthe microheterogeneity of

proteins

- Usefulfor separatingthe isoenzymes.

- Research inenzymology, immunology

- Forensic, foodandagriculture industry

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 Two-dimensional polyacrylamide
gel electrophoresis

Principle:

Second dimensionby Technique combineswith

SDS-PAGE IEFasfirstdimensional.
• Separateaccording molecular • Whichseparate accordingto
size. the charge.

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 This combinationgives sophisticated analytical method
for analysing the protein mixture.
 Size very from 20*20cm to the minigel.
 IEF is carried on acrylamide gel (18cm*3mm), with
8M urea.
 After separation, placed on 10% SDS-PAGE for further
separation .
 Usedin field of proteomics.
 Canseparate1000 to 3000 proteins from the cell or tissueextract.

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69
 Isotachophoresis
 Used for separation of smaller ionic substances.
 They migrate adjacent in contact with one another, but not
overlapping.
 The sample is not mixed with the buffer prior to run.
 Hence current flow is carried entirely by the sample ions.
 Faster moving ions migrate first and the adjacent ones next
with no gap between the zone .
 Allionsmigrate at the rate of fastest ion in zones.
Then it ismeasuredbyUVabsorbance.
Application-
Separationofsmallanionsand cations
Aminoacids
Peptides
Nucleotides
Nucleosides
Proteins.

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 Pulsed-Field Electrophoresis
Powerisappliedalternativelyto different pair of electrodes

Electrophoretic fieldiscycledat 105-1800

Becauseof whichthe moleculehaveto orient to the new field direction
Thispermit separation of largemoleculelike DNA.

Applied:for typingvariousstrains of DNA.

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 Immuno electrophoresis
 Immunoelectrophoresis refers to precipitation in agar under an
electric field.

 It is a process of combination of immuno-diffusion and

electrophoresis.

 An antigen mixture is first separated into its component parts by

electrophoresis and then tested by double immuno-diffusion.

 Antigens are placed into wells cut in a gel (without antibody) and
electrophoresed. A trough is then cut in the gel into which
antibodies are placed.

 The antibodies diffuse laterally to meet diffusing antigen, and

lattice formation and precipitation occur permitting determination
of the nature of the antigens.

 The term “immunoelectrophoresis” was first coined by Grabar and

Williams in 1953.
 Principle

When electric current is applied to a slide layered with

gel, antigen mixture placed in wells is separated into
individual antigen components according to their
charge and size. Following electrophoresis, the
separated antigens are reacted with specific antisera
placed in troughs parallel to the electrophoretic
migration and diffusion is allowed to occur. Antiserum
present in the trough moves toward the antigen
components resulting in formation of separate
precipitin lines in 18-24 hrs, each indicating reaction
between individual proteins with its antibody.
-Precipitation reactions are based on the interaction of antibodies and antigens. They
are based on two soluble reactants that come together to make one insoluble
product, the precipitate (Figure).

-These reactions depend on the formation of lattices (cross-links) when antigen and
antibody exist in optimal proportions.[ it is known aszone ofequivalence and appears
to us asprecipitation].

-Excessof either component reduces lattice formation and subsequentprecipitation.

 Procedure
Agarose gel is prepared on a glass slide put in a horizontal position.
Using sample template, wells are borne on the application zone carefully.
The sample is diluted 2:3 with protein diluent solution (20μl antigen solution
+10 μl diluent).
Using a 5 μl pipette, 5 μl of control and sample is applied across each
corresponding slit (Control slit and Sample slit).
The gel is placed into the electrophoresis chamber with the samples on the
cathode side, and electrophoresis run for 20 mins/ 100 volts.
After electrophoresis completes, 20 μl of the corresponding antiserum is added
to troughs in moist chamber and incubated for 18- 20 hours at room
temperature on a horizontal position.
The agarose gel is placed on a horizontal position, and dried with blotter
sheets.
The gel in saline solution is soaked for 10 minutes and the drying and washing
repeated twice again.
The gel is dried at a temperature less than 70°C and may be stained with
 Results

o Presence of elliptical precipitin arcs represents antigen-

antibody interaction.

o Absence of formation of precipitate suggests no

reaction.

o Different antigens (proteins) can be identified based on

the intensity, shape, and position of the precipitation
lines.
 Applications

The test helps in identification and approximate quantization of

various antigens in immunology.

suspected monoclonal and polyclonal gammopathies.

Used to analyze complex protein mixtures containing different

antigens.

The medical diagnostic use is of value where certain proteins are

suspected of being absent (e.g., hypogammaglobulinemia) or
overproduced (e.g., multiple myeloma).

This method is useful to monitor antigen and antigen-antibody

purity and to identify a single antigen in a mixture ofantigens.
 Capillary electrophoresis
oTechnique first described by- Jorgensen and Lukas (1980’s). As the
name suggest, the separation is carried in a narrow bore Capillary

50μm – ID.
300 μm –ED.
Length – 50-100cm.
Fused silica capillary tube.

Polyimide coatingexternal.

Packedwith the bufferin use.

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 Components:

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 Sampleapplication isdone byeither ofone method

Highvoltage Pressure
injection injection

High voltageinjection Pressure injection

The buffer reservoir is Anodic end of capillary is
replaced by the sample removed from buffer and
reservoir the high placed in air tight sample
voltage is applied (+ sol with pressuresample
electrode) buffer is pushed intocapillary
reservoir is placed again kept back in the
andvoltageappliedfor the buffersampleandvoltage
separation. is applied.
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  High voltage is applied (up to 50 kV)

 The components migrate at different rate along the length.

 Although separated by the electrophoretic migration, all the
sample is drawn towards cathode by electroendosmosis.

 Since this flow is strong, the rate of electro endosmotic flow is

greater than the Electrophoretic velocity of the analyte ion,
regardless of the charge.

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 Positively charged molecule reach the cathode first
(electrophoretic migration+ electro osmoticflow).

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DETECTION:

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 Troubleshooting :
Adsorption of protein to the wall of capillary – leading to
smearing of protein – viewed as peak broadening – or
complete loss of protein.

- Use of neutral coating group to the inner surface of

the capillary.

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 Multiple myelomatesting

Haemoglobinopathy
clinical screening.
applications
include HbA1c

Monitoringchronic
alcoholism(GGT).
 Advantage over conventional

• Online detection.

• Improved quantification.

• Almost complete automation.

• Reduced analysis time.

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 Microchipelectrophoresis
Current advanced method.
Development in technique include
Integrated microchip design
Advanced detection system

New application

Protein and DNA separation can be

done

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 Instrumentation

Similar to thecapillary electrophoresis.

Separationchannel
Sample injection(50-100pL)
Reservoirs
Voltage(1-4kV)
samplepreparation
Pre column or post column reactors.
Classical Cross-T design.
Time periodof 50-200sec.

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 Detector :
Laser inducedfluorescence

Electrochemical detectors

Pulsed amperometricdetector

Sinusoidalvoltametry

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 References

Tietz- Textbookofclinicalchemistry.

Kaplan- clinicalchemistry.

YouTube and Google images.

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THANK
YOU