Electrophoresis complete

Dr. Anurag Yadav

Post-graduate, Biochemistry

Father Muller Medical college

ELECTROPHORESIS

1 Dr Anurag yadav,Bio-FMMC

CONTENT

Dr Anurag yadav,Bio-FMMC 2

Introduction

Principle

Factors affecting

Conventional electrophoresis

General operation

Technical and practical Consideration

Types of electrophoresis

INTRODUCTION


Electrophoresis is the migration of charged particles or

molecules in a medium under the influence of an applied

electric field.

Wallach's Interpretation of Diagnostic Tests

Electrophoresis


a separation technique

Simple, rapid and highly sensitive

used in clinical laboratories to separate charged molecules from each

other in presence of electric field

– Proteins in body fluids: serum, urine, CSF

– Proteins in erythrocytes: hemoglobin

– Nucleic acids: DNA, RNA

Clinical applications of Electrophoresis

Serum Protein Electrophoresis

Lipoprotein Analysis

Diagnosis of Haemoglobinopathies and Haemoglobin A1c

Determination of Serum Protein Phenotypes and Micro

heterogeneities eg. α1- antitrypsin deficiency, MM

Genotyping of Proteins eg. ApoE analysis for Alzheimer’s disease

(polymorphic protein)

Small Molecules (Drugs, Steroids) Monitoring

Cerebrospinal Fluid Analysis

Urine Analysis ( determination of GNs)

Principle :


Comprehensive term that refers to the migration of charged particle of

any size in liquid medium under the influence of an electric field.

Depending on kind of charge the molecule carry, they move towards

either

To cathode

Or to Anode

An ampholyte become positively charged in acidic condition and migrate

to cathode, in alkaline condition they become negatively charge and

migrate to anode.


Eg: as protein contain the ionizable amino and carboxyl

group.

The rate of migration of an ion in electrical field depend on

factors,

1. Net charge of molecule

2. Size and shape of particle

3. Strength of electrical field

4. Properties of supporting medium

5. Temperature of operation

1. Mobility


Under the electrical field, the mobility of the particle is

determined by two factors:

Its charge

Frictional coefficient

Size and shape of the particle decide the velocity with which the

particle will migrate under the given electrical field and the

medium.


2. Strength of electrical field


It determined by the force exerted on the particle, and the charge the particle

carrying.

F=QV

when force is exerted on the particle it start moving, however the moment is

restricted by the experience of the frictional force because of the viscosity.

Effect of pH on Mobility


As the molecule exist as amphoteric , they will carry the

charges based on the solvent pH.

Their overall net charge is NEUTRAL when it is at zwitter

ion state. And hence the mobility is retarded to zero.

Mobility is directly proportional to the magnitude of the

charge, which is functional of the pH of solvent.

The pH is maintained by the use of Buffers of different pH.

Factors Affecting Electrophoresis

Conventional electrophoresis


Instrumentation :

Two reservoir for the buffer

Power supply and Electrodes

Separation medium

Power supply


Drives the moment of ionic species in the medium and allow

the adjustment and control of the current or voltage.

Constant delivery is required.

Pulsed power can also be applied.

Buffer


The buffer in electrophoresis has twofold purpose:

Carry applied electrical current

They set the pH as which electrophoresis is carried out.

Thus they determine;

Type of charge on solute.

Extent of ionization of solute

Electrode towards which the solute will migrate.

The buffer ionic strength will determine the thickness of the ionic

cloud.

Commonly buffers used;


Buffer

pH value

Phosphate buffer

around 7.0

Tris-Borate-EDTA buffer (TBE)

around 8.0

Tris-Acetate EDTA buffer (TAE)

above 8.0

Tris Glycine buffer (TG)

more than 8.5

Tris -Citrate-EDTA buffer (TCE)

around 7.0

Tris -EDTA buffer (TE)

around 8.0

Tris -Maleic acid -EDTA buffer (TME)

around 7.5

Lithium Borate - buffer (LB)

around 8.6

Supporting medium


Supporting medium is an 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 to the charge to mass ratio of protein

depending on the pore size of the medium, possibly the

molecular size.

Chemical nature

inert

Availability

easy

Electrical conductivity

high

Adsorptivity

low

Sieving effect

desirable

Porosity

controlled

Transparency

high

Electro-endosmosis (EEO)

low

Rigidity

moderate to high

Preservation

feasible

Toxicity

low

Preparation

easy

Properties:


- Starch gel

- Cellulose acetate

- Agarose

- Polyacrylamide gel

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 agarose.


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.

APPLICATION:

Widely used in Immuno

electrophoresis.

Gel Structure of Agarose:

Cellulose acetate


Thermoplastic resin made by treating cellulose with acetic

anhydride to acetylate the hydroxyl group.

When dry, membrane contain about 80% air space within fibers

and brittle film.

As the film is soak in buffer, the space are filled.

Because of their opacity, the film has to be made transparent by

soaking in 95:5 methanol:glacial acetic acid.

It can be stored for longer duration.

Polyacrylamide


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.

They are defined in terms of total percentage of acrylamide present, and

pore size vary with conc.


Made in conc between 3-30% acrylamide.

Thus low % has large pore size and vice versa.

Proteins are separated on the basis of charge to mass ratio and

molecular size, a phenomenon called Molecular sieving.

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.

General Operation


The general operation of the conventional electrophoresis

include;

Separation

Detection

Quantification

a. Electrophoresis Separation


When performed on precast or agarose gel, following steps

are followed;

- Excess buffer removed

- 5-7 μL sample

- Placed in electrode chamber

- Current application

- Gel is rinsed, fixed and dried

- Stained

- Scanned under densitometry

b. Staining


Protein is ppt in gel by using acetic acid or methanol

(this will prevent diffusion of protein out of the gel when

submerged in stain solution)

Amount of dye taken by sample is affected by many factors,

Type of protein

Degree of denaturation

Different stains of Electrophoresis

Plasma Proteins

- Amido black

- Coomassie Brilliant Blue

- Bromophenol Blue

Hemoglobins

- Amido black

- Coomassie Brilliant Blue

- Ponceau Red

Lipoproteins

- Sudan Black

DNA ( Fluorescent dyes)

- Ethidium Bromide

- Sybr Green, Sybr Gold

Staining Systems

Proteins

General – Coomassie brilliant blue R, Kenacid blue, Amido

black.

Specific – Oil red O, PAS, Rubeanic acid, Transferrin-specific & for calcium binding proteins

Steps * fixing

* staining

* destaining

Allozymes - Histochemical staining

DNA - EtBr, SyBR green, Propidium iodide and silver staining

C. Detection and Quantification


Once separated, protein may be detected by staining

followed by the quantification using the densitometer or by

direct measuring using an optical detection system under set

at 210nm.

Separation type Wavelength

Serum protein 520-640nm

Isoenzymes 570nm

Lipoproteins 540-600nm

DNA fragments 254-590nm

CSF protein ----

The selection of the wavelength is the property o type of stain used for the identification of

separation.

Few technical considerations

What is EEO & why low???

Common effect of variables on

separation


pH Changes charge of analyte, effective mobility; structure of analyte-

denaturing or dissociating a protein.

Ionic strength Changes in voltage; increased ionic strength reduces migration velocity

and increase heating.

Ions present Change migration speed; cause tailing of bands.

Current Too high current cause overheating.

Temperature Overheating cause denature protein; lower temp reduce diffusion but also

migration; there is no effect on resolution.

Time Separation of bands increases linearly with time, but dilution of bands

increase with square root of time.

Medium Major factors are endosmosis and pore size effect, which effect migration

velocities.

TYPES OF ELECTROPHORESIS

1) Zone Electrophoresis a) Paper Electrophoresis

b) Gel Electrophoresis

c) Thin Layer Electrophoresis

d) Cellulose acetate Electrophoresis

2) Moving Boundary Electrophoresis

a) Capillary Electrophoresis

b) Isotachophoresis

c) Isoelectric Focussing

d) Immuno Electrophoresis

39

CLASSIFICATION

• Traditional methods, using a rectangular gel regardless of thickness

Slab gel electrophoresis

• DISContinuities in electrophoretic matrix caused by layers of polyacrylamide/starch gel that differ in composition & pore size

Disc electrophoresis

CLASSIFICATION

• IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient

Isoelectric focusing

electrophoresis

• Completely separates smaller ionic substances into adjacent zones tat contact one another with no overlap & all migrate at the same rate.

Isotachophoresis

CLASSIFICATION

• Power is alternately applied to different pair of electrodes/ electrode arrays, so the electrophoretic field is cycled b/w 2 directions.

Pulse-Field electrophoresis

• Charge-dependent IEP in the first dimension.

• Molecular weight dependent electrophoresis in second.

2-D electrophoresis

SUPPORT MEDIA IN SEPERATION

Molecular size

• Gradient gels

• Gels containing denaturants

Molecular size & Charge

• Gel electrophoresis

• Immunoelectrophoresis

• 2D electrophoresis

ENHANCED-RESOLUTION TECHNIQUES:

• Isotachophoresis

• Disk electrophoresis

• Isoelectric focusing

CLASSIFICATION

Types

Horizontal

Vertical

CLASSIFICATION

Moving boundary electrophoresis

Zone electrophoresis

Cellulose acetate electrophoresis


Although older, still has number of application.

Has advantage over paper, being homogenous medium with

uniform pore size and doesnot absorb the protein.

Much less tailing of the band.

Resolution is better than paper.


Much simpler to run. Can be used as single sample or

multiple sample run.

Acetate paper is first wetted in the buffer, and the sample is

loaded.

The strip is kept for the electrophoretic run.

6-8 V/cm for about 3 hr.

The protein separation is stained, for better visualization.


Although used for the serum protein separation, but replaced

by the agarose gel ( which give better resolution).

The enzymes can easily detected by using Zymogram

technique.

Cellulose Acetate Electrophoresis:

Application:

• Serum protein electrophoresis

• Hemoglobin electrophoresis

• Lipoprotein electrophoresis

• Enzymes (zymogen technique)

• ALP isoenzyme electrophoresis


Better Resolution.


• Resolution less as compared to PAGE

• 8-9 serum fractions as compared to 30 with disk/PAGE

DISADVANTAGES

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.


Mercaptoethanol will break the disulphide bridges.

SDS binds strongly to and denatures the protein.

Each protein is fully denatured and open into rod-shape with series of negatively charged SDS molecule on polypeptide chain.

SDS is an anionic detergent.

The sample is first boiled for 5min in buffer containing

• Beta-Mercaptoethanol

• SDS


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.

Also referred as Discontinuous gel electrophoresis.

Components



Stacking gel: ordering/arranging and conc the macromolecule before entering the field of separation. (4% of acrylamide)

• Purpose is to concentrate protein sample in sharp band before enters main separating gel.

Running gel: the actual zone of separation of the particle/molecules based on their mobility. (15% of acrylamide)

Pore size: routinely used as 3% to 30% which is of pore size 0.2nm to 0.5nm resp.


Movement of particle


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



In separating gel, protein separate owing to molecular sieving

properties.

Smaller proteins pass more easily, larger one retarded by

friction.

- Research tool

- Measuring molecular weight

- Peptide mapping

- Protein identification

- Determination of sample purity

- Identifying disulfide bonds

- Separation of proteins and establishing size

- Blotting

- Smaller fragments of DNA

- Separation of nucleic acids

- Major clinical use – ALP separation

APPLICATION:

ADVANTAGES:

- Clear, fairly easy to prepare

- Exhibit reasonable mechanical strength over acrylamide conc

- Low endosmosis effect

DISADVANTAGES

- Gel preparation and casting- exacting n time-consuming

- Complete reproducibility of gel preparation not possible

STAINING:

Fluorescent stains - Ethidium bromide – Nucleic acids

Silver stain for protein gel (sensitive 50 times dye based)

Dye based – Coomassie blue – 50ng protein band

Tracking dyes – BPB> xylene cyanol, Orange G

Native (buffer) gel


Done by using the polyacrylamide gel (7.5%).

As used for the enzyme separation, the denaturing agent is

not added - hence SDS is absent.

pH of 8.7

Proteins are separated according to the electrophoretic

mobility & Sieving effect of the gel.


Alternative approach for enzyme detection is to include the

substrate on agarose gel, which is poured over acrylamide gel.

The diffusion and interaction of the substrate and the enzyme

results in color formation.

This can be cut and used for

Total protein estimation

Enzyme activity.

Gradient gel


This is again an polyacrylamide gel system.

Instead of running a slab of uniform pore size, a gradient gel

is formed.

Uniformly from 5% to 25% acrylamide from top to bottom.

The highest conc gradient is layed first and than decreasing

gradient is poured.

But the sample move down, were the pore size reduces along

the path.


Normally run with the stacking gel at the top.

Advantage :

Greater range of protein can be separated. (Complex mixtures

can be run.)

Protein with similar molecular range may be resolved.

Protein moves till the pore size become smaller n limit its

descend further.

Proteins separated will have a distinct sharp bands.

Isoelectric focussing gels


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 little 0.01 of pH unit.

Most widely used as the horizontal gel slab.


Different gradient of the pH along

the length of the separating gel.

Establishment of ph gradient:


This is achieved by the ampholyte & must have following prop:

Must dictate pH course (buffering capacity at their Ip)

Should have conductance at their Ip.

Low molecular weight

Soluble in water

Low light absorbance at 280nm.

Available commercially with pH band (3-11)

Eg: Ampholine, Pharmalyte and Bio-lyte.


Movement



Duration : 2-3h

High voltage : 2500V

Cooling plates : 100C

Stable power pack

Fixing (trichloroacetic acid) and Staining (Coomassie

Brilliant blue)


Application:

- Highly sensitive for studying the microheterogeneity of

proteins

- Useful for separating the isoenzymes.

- Human genetic lab

- Research in enzymology, immunology,

- Forensic, food and agriculture industry,

Two-dimensional polyacrylamide gel electrophoresis


Principle :

Technique combines with IEF as first dimensional.

• Which separate according to the charge.

Second dimension by SDS-PAGE

• Separate according molecular size.


Thus combination gives sophisticated

analytical method for analysing the

protein mixture.

Size very from 20*20cm to the minigel.

IFE is carried on acrylamide gel

(18cm*3mm), with 8M urea.

After separation, placed on 10% SDS-

PAGE for further separation .


Used in field of proteomics.

Can separate 1000 to 3000 proteins from the cell or an tissue

extract.


Isotachophoresis


Used for separation of smaller ionic substances.

They migrate adjacent with contact 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 .


All ions migrate at the rate of fastest ion in zones.

Then it is measured by UV absorbance.

Application-

Separation of small anions and cations

Amino acids

Peptides

Nucleotides

Nucleosides

Proteins.

Pulsed-Field Electrophoresis


Power is applied alternatively to different pair

of electrodes

Electrophoretic field is cycled at 105-1800

Because of which the molecule have to orient

to the new field direction

This permit separation of large molecule like

DNA .

Applied: for typing various strain DNA.

High voltage electrophoresis


First described by Michl.

As the name describe, the electrophoresis is carried under

the very high voltage.

This is required for the substances of lower molecular weight

which will have considerable high diffusion rate.

Eg: amino acids, peptides.


The voltage applied was ranging from

2500-10000 V or

50-200V/cm, 500mA.

This resulted in better resolution and even very rapid

separation.

And even with tremendous amount of heat generation.

To tackle this, it need a good cooling system.

components


Buffer reservoirs

Cooling plate

Pressure pad

electrodes

Power source

Insulated cover

Wick

Refrigerating unit


Precautions :Temperature of the system has to be maintained

constant.

Plate dimension 50*50cm

The HVE one direction can be combined with the

chromatography- which is right angle to first.

Possible even to run in two direction at two different pH.

Capillary electrophoresis


Technique first described by- Jorgensen and Lukacs (1980’s)

Also referred as

High performance capillary electrophoresis(HPCE)

Capillary zone electrophoresis (CZE)

Free solution capillary electrophoresis (FSCE)

Capillary electrophoresis (CE)


The sensitivity has made it as one of the choice for many

biomedical and clinical analyses.

Application : used to separate

Amino acids

Peptides Proteins DNA

fragments Nucleic

acid

Drugs / even

metals.


Other clinical applications

include

Multiple myeloma testing (6bands).

Haemoglobinopathy screening.

HbA1c

Monitoring chronic alcoholism (GGT).

Components :



Small amount of sample is required (5-

30 μm3)

Introduced into the capillary with

appropriate buffer at anode end.

High voltage injection Pressure injection


The buffer reservoir is

replaced by the sample

reservoir the high

voltage is applied (+

electrode) buffer

reservoir is placed again

and voltage applied for the

separation.

Anodic end of capillary is

removed from buffer and

placed in air tight sample

sol with pressure sample

is pushed into capillary

kept back in the

buffer sample and voltage

is applied.

Sample application is done by either of one method

High voltage injection

Pressure injection


50μm – ID.

300 μm – ED.

Length – 50-100cm.

Fused silica capillary tube.

Polyimide coating external.

Packed with the buffer in use.

As the name suggest, the separation is carried in a narrow

bore Capillary


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 electroendosmotic

flow is greater than the electrophoretic velocity of the

analyte ion, regardless of the charge.


Positively charged molecule reach the cathode first (electrophoretic migration + electroosmotic flow).


DETECTION:

near to cathode end,

viewing window

- Detected by the

ultraviolet monitor,

transmit signal and

integrated by

computer.

- Refractive index

- Fluorescence

- CE-MS


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.


Advantage over slab type:

Reduce the problem of heating effect.

Large surface to volume ratio.

Less diffusion of the separated bands.


Variations in technique:

Add of surfactant to buffer i.e., SDS (for Neutral molecules).

Micellar formation In MECC- electrophoresis + chromatography.

Different modes of operation


Capillary zone electrophoresis :

- Separation principle based on charge to mass ratio of

molecule.

- Separation is faster.

- Due to High EOF, the molecules regardless of the charge,

they are moved to cathode.



Micellar electrokinetic chromatography:

- It is an hybrid.

- Used for separation of the neutral and charged solutes.

- The separation is accomplished by micelles formation. (8-

9mmol/L for SDS)

- During migration, micelle interact with analyte as

chromatographic manner and the separation is brought

about.




Capillary gel electrophoresis:

- Identical to the slab.

- Separation based on the sieving.

- The capillary is filled with “sieving matrix” or “soluble

polymer network”.

- Low viscosity, self entangling for formation of pore size.

- Variety of polymeric matrices are available for DNA and Protein.

- Cross linked polyacrylamide- choice of polymer.


Advantage over conventional

• Online detection.

• Improved quantification.

• Almost complete automation.

• Reduced analysis time.

• Wider choice of gel matrices.

• Linear polyacrylamide, derivative of cellulose, galactomannan, glucomannan, polyvinyl alcohol, polyethyleneoxide, agarose, dextran, polymethylacrylamide, and polyacryloylethoxyethenol.



Capillary isoelectric Focussing Electrophoresis:

- Is comparable to tube IEF.

- Carried out in the capillary.

- The focused zone migrate to the detector with the separated

sample.

- cIEF is completed in ~15 min.



Capillary Isotachophoresis:

- Same feature as ITP.

- Except condition of pure ITP not achieved.

- Typically used for online sample preconcentration.

- CZE, MEKC, CGE.


a. Capillary Isotachophoresis

b. Capillary gel electrophoresis

c. Capillary isoelectric Focussing Electrophoresis

d. Micellar electrokinetic chromatography

summary

Capillary Electrophoresis (CE) versus High Performance Liquid Chromatography (HPLC)


CE has flat flow, compared to pumped parabolic flow of HPLC.

Flat flow will have narrower peaks & better resolution.

CE has greater peak capacity.


HPLC is more thoroughly developed.

HPLC is more complex than CE.

HPLC has wider variety of column length and packing

Both techniques uses similar modes of detection.

Can be used complementary to one another.


Microchip electrophoresis


Current advanced method.

Development in technique include

Integrated microchip design

Advanced detection system

New application

Protein and DNA separation can be done

Instrumentation


Similar to the capillary electrophoresis.

Separation channel

Sample injection (50-100pL)

Reservoirs

Voltage (1-4kV)

sample preparation

Precolumn or postcolumn reactors.

Classical Cross-T design.

Time period of 50-200sec.


Detector :

Laser induced fluorescence

Electrochemical detectors

Pulsed amperometric detector

Sinusoidal voltametry

Application


An alternative for the DNA analysis.

Herpes simplex virus DNA in CSF for diagnosing encephalitis.

Gene rearrangement correlative with lymphoproliferative

disorders.

Polymorphisms in gene.

Tetranucleotide associated with hypercholesterolemia.

Diagnosing fragile X syndrome.

Muscular dystrophy.

Anthracis specific PCR product.


References


Keith Wilson- Principles and techniques of biochemistry

and molecular biology.

Upadhyay- biophysical chemistry.

Tietz- Text book of clinical chemistry.

Kaplan- clinical chemistry.

YouTube and Google images.

Health & Medicine

Electrophoresis complete