Electrophoresis by Dr. Anurag Yadav

electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis

1. Dr. Anurag Yadav
Post-graduate, Biochemistry
Father Muller Medical college
ELECTROPHORESIS
1
Dr Anurag yadav,Bio-FMMC

2. CONTENT
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Introduction
Principle
Factors affecting
Conventional electrophoresis
General operation
Technical and practical Consideration
Types of electrophoresis

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

4. Wallach's Interpretation of Diagnostic Tests

5. Electrophoresis
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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

6. 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)

7. Principle :
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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.

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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

9. 1. Mobility
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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.

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11. 2. Strength of electrical field
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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.

12. Effect of pH on Mobility
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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.

13. Factors Affecting Electrophoresis

14. Conventional electrophoresis
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Instrumentation :
Two reservoir for the buffer
Power supply and Electrodes
Separation medium

15. Power supply
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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.

16. Buffer
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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.

17. Commonly buffers used;
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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

18. Supporting medium
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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.

19. 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:

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-Starch gel
-Cellulose acetate
-Agarose
-Polyacrylamide gel

21. Agarose Gel
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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.

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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:

23. Cellulose acetate
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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.

24. Polyacrylamide
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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.

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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.

26. General Operation
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The general operation of the conventional electrophoresis
include;
Separation
Detection
Quantification

27. a. Electrophoresis Separation
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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

28. b. Staining
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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

29. 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

30. 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

31. C. Detection and Quantification
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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.

32. Few technical considerations

33. What is EEO & why low???

38. Common effect of variables on
separation
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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.

39. 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
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40. 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

41. 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

42. 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

43. SUPPORT MEDIA IN SEPERATION
Molecular size
•Gradient gels
•Gels containing denaturants
Molecular size &
Charge
•Gel electrophoresis
•Immunoelectrophoresis
•2D electrophoresis

44. ENHANCED-
RESOLUTION
TECHNIQUES:
•Isotachophoresis
•Disk electrophoresis
•Isoelectric focusing

45. CLASSIFICATION
Types
Horizontal
Vertical

46. CLASSIFICATION
Moving
boundary
electrophoresis
Zone
electrophoresis

47. Cellulose acetate electrophoresis
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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.

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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.

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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.

50. Cellulose Acetate Electrophoresis:
Application:
•Serum protein
electrophoresis
•Hemoglobin electrophoresis
•Lipoprotein electrophoresis
•Enzymes (zymogen
technique)
•ALP isoenzyme
electrophoresis

51. Cellulose Acetate Electrophoresis:
Better Resolution.

52. Cellulose Acetate Electrophoresis:
•Resolution less as
compared to PAGE
•8-9 serum fractions as
compared to 30 with
disk/PAGE
DISADVANTAGES

53. SDS-PAGE
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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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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

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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.
Also referred as Discontinuous gel electrophoresis.

56. Components
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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.

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59. Movement of particle
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[Cl] > [protein-SDS] > [Glycinate]

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In separating gel, protein separate owing to molecular sieving
properties.
Smaller proteins pass more easily, larger one retarded by
friction.

62. -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:

63. 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

64. 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

65. Native (buffer) gel
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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.

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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.

67. Gradient gel
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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.

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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.

69. Isoelectric focussing gels
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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.

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Different gradient of the pH along
the length of the separating gel.

71. Establishment of ph gradient:
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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.

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73. Movement
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Duration : 2-3h
High voltage : 2500V
Cooling plates : 10
0
C
Stable power pack
Fixing (trichloroacetic acid) and Staining (Coomassie
Brilliant blue)

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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,

76. Two-dimensional polyacrylamide
gel electrophoresis
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Principle :
Technique combines with
IEF as first dimensional.
•Which separate according to
the charge.
Second dimension by
SDS-PAGE
•Separate according molecular
size.

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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 .

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Used in field of proteomics.
Can separate 1000 to 3000 proteins from the cell or an tissue
extract.

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80. Isotachophoresis
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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 .

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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.

82. Pulsed-Field Electrophoresis
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Power is applied alternatively to different pair
of electrodes
Electrophoretic field is cycled at 105-180
0
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.

83. High voltage electrophoresis
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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.

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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.

85. components
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Buffer reservoirs
Cooling plate
Pressure pad
electrodes
Power source
Insulated cover
Wick
Refrigerating unit

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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.

87. Capillary electrophoresis
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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)

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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.

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Other clinical
applications
include
Multiple myeloma testing
(6bands).
Haemoglobinopathy
screening.
HbA1c
Monitoring chronic
alcoholism (GGT).

90. Components :
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91. Dr Anurag yadav,Bio-FMMC
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Small amount of
sample is required (5-
30 μm
3
)
Introduced into the
capillary with
appropriate buffer at
anode end.

92. High voltage injection Pressure injection
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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

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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

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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 electroendosmotic
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 + electroosmotic flow).

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DETECTION:
near to cathode end,
viewing window
- Detected by the
ultraviolet monitor,
transmit signal and
integrated by
computer.
-Refractive index
-Fluorescence
-CE-MS

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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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Advantage over slab type:
Reduce the problem of heating effect.
Large surface to volume ratio.
Less diffusion of the separated bands.

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Variations in technique:
Add of surfactant to buffer i.e., SDS (for Neutral molecules).
Micellar formation In MECC- electrophoresis + chromatography.

100. Different modes of operation
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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.

101. Different modes of operation
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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.

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103. Different modes of operation
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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.

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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.

105. Different modes of operation
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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.

106. Different modes of operation
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Capillary Isotachophoresis:
-Same feature as ITP.
-Except condition of pure ITP not achieved.
-Typically used for online sample preconcentration.
-CZE, MEKC, CGE.

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a. Capillary Isotachophoresis
b. Capillary gel electrophoresis
c. Capillary isoelectric Focussing
Electrophoresis
d. Micellar electrokinetic
chromatography
summary

108. Capillary Electrophoresis (CE) versus High
Performance Liquid Chromatography (HPLC)
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CE has flat flow, compared to pumped parabolic flow of HPLC.
Flat flow will have narrower peaks & better resolution.
CE has greater peak capacity.

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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.

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111. Microchip electrophoresis
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Current advanced method.
Development in technique include
Integrated microchip design
Advanced detection system
New application
Protein and DNA separation can be done

112. Instrumentation
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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.

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Detector :
Laser induced fluorescence
Electrochemical detectors
Pulsed amperometric detector
Sinusoidal voltametry

114. Application
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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.

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116. References
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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.