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Dr.AnuragYadav
Post-graduate, Biochemistry
Father Muller Medical college
ELECTROPHORESIS
1 DrAnurag yadav,Bio-FMMC
CONTENT
DrAnurag yadav,Bio-FMMC2
 Introduction
 Principle
 Factors affecting
 Conventional electrophoresis
 General operation
 Technical and practical Consideration
 Types of electrophoresis
INTRODUCTION
DrAnurag yadav,Bio-FMMC3
 Electrophoresis is the migration of charged particles or
molecules in a medium under the influence of an applied
electric field.
Wallach's Interpretation of DiagnosticTests
Electrophoresis
DrAnurag yadav,Bio-FMMC5
 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
 LipoproteinAnalysis
 Diagnosis of Haemoglobinopathies and HaemoglobinA1c
 Determination of Serum Protein Phenotypes and Micro
heterogeneities eg. α1- antitrypsin deficiency, MM
 Genotyping of Proteins eg.ApoE analysis forAlzheimer’s disease
(polymorphic protein)
 Small Molecules (Drugs, Steroids) Monitoring
 Cerebrospinal FluidAnalysis
 UrineAnalysis ( determination of GNs)
Principle :
DrAnurag yadav,Bio-FMMC7
 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 toAnode
 An ampholyte become positively charged in acidic condition and migrate
to cathode, in alkaline condition they become negatively charge and
migrate to anode.
DrAnurag yadav,Bio-FMMC8
 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
DrAnurag yadav,Bio-FMMC9
 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.
DrAnurag yadav,Bio-FMMC10
2. Strength of electrical field
DrAnurag yadav,Bio-FMMC11
 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
DrAnurag yadav,Bio-FMMC12
 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
DrAnurag yadav,Bio-FMMC14
 Instrumentation :
 Two reservoir for the buffer
 Power supply and Electrodes
 Separation medium
Power supply
DrAnurag yadav,Bio-FMMC15
 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
DrAnurag yadav,Bio-FMMC16
 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;
DrAnurag yadav,Bio-FMMC17
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
DrAnurag yadav,Bio-FMMC18
 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:
DrAnurag yadav,Bio-FMMC20
- Starch gel
- Cellulose acetate
- Agarose
- Polyacrylamide gel
Agarose Gel
DrAnurag yadav,Bio-FMMC21
 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.
DrAnurag yadav,Bio-FMMC22
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
DrAnurag yadav,Bio-FMMC23
 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
DrAnurag yadav,Bio-FMMC24
 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.
DrAnurag yadav,Bio-FMMC25
 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
DrAnurag yadav,Bio-FMMC26
 The general operation of the conventional electrophoresis
include;
Separation
Detection
Quantification
a. Electrophoresis Separation
DrAnurag yadav,Bio-FMMC27
 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
DrAnurag yadav,Bio-FMMC28
 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
DrAnurag yadav,Bio-FMMC31
 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???
Electrophoresis principle and types by Dr. Anurag Yadav
Electrophoresis principle and types by Dr. Anurag Yadav
Electrophoresis principle and types by Dr. Anurag Yadav
Electrophoresis principle and types by Dr. Anurag Yadav
Common effect of variables on
separation
DrAnurag yadav,Bio-FMMC38
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
DrAnurag yadav,Bio-FMMC47
References
DrAnurag yadav,Bio-FMMC48
 KeithWilson- Principles and techniques of biochemistry
and molecular biology.
 Upadhyay- biophysical chemistry.
 Tietz-Text book of clinical chemistry.
 Kaplan- clinical chemistry.
 YouTube and Google images.

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Electrophoresis principle and types by Dr. Anurag Yadav

  • 1. Dr.AnuragYadav Post-graduate, Biochemistry Father Muller Medical college ELECTROPHORESIS 1 DrAnurag yadav,Bio-FMMC
  • 2. CONTENT DrAnurag yadav,Bio-FMMC2  Introduction  Principle  Factors affecting  Conventional electrophoresis  General operation  Technical and practical Consideration  Types of electrophoresis
  • 3. INTRODUCTION DrAnurag yadav,Bio-FMMC3  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 DiagnosticTests
  • 5. Electrophoresis DrAnurag yadav,Bio-FMMC5  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  LipoproteinAnalysis  Diagnosis of Haemoglobinopathies and HaemoglobinA1c  Determination of Serum Protein Phenotypes and Micro heterogeneities eg. α1- antitrypsin deficiency, MM  Genotyping of Proteins eg.ApoE analysis forAlzheimer’s disease (polymorphic protein)  Small Molecules (Drugs, Steroids) Monitoring  Cerebrospinal FluidAnalysis  UrineAnalysis ( determination of GNs)
  • 7. Principle : DrAnurag yadav,Bio-FMMC7  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 toAnode  An ampholyte become positively charged in acidic condition and migrate to cathode, in alkaline condition they become negatively charge and migrate to anode.
  • 8. DrAnurag yadav,Bio-FMMC8  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 DrAnurag yadav,Bio-FMMC9  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.
  • 11. 2. Strength of electrical field DrAnurag yadav,Bio-FMMC11  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 DrAnurag yadav,Bio-FMMC12  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.
  • 14. Conventional electrophoresis DrAnurag yadav,Bio-FMMC14  Instrumentation :  Two reservoir for the buffer  Power supply and Electrodes  Separation medium
  • 15. Power supply DrAnurag yadav,Bio-FMMC15  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 DrAnurag yadav,Bio-FMMC16  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; DrAnurag yadav,Bio-FMMC17 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 DrAnurag yadav,Bio-FMMC18  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:
  • 20. DrAnurag yadav,Bio-FMMC20 - Starch gel - Cellulose acetate - Agarose - Polyacrylamide gel
  • 21. Agarose Gel DrAnurag yadav,Bio-FMMC21  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.
  • 22. DrAnurag yadav,Bio-FMMC22 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 DrAnurag yadav,Bio-FMMC23  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 DrAnurag yadav,Bio-FMMC24  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.
  • 25. DrAnurag yadav,Bio-FMMC25  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 DrAnurag yadav,Bio-FMMC26  The general operation of the conventional electrophoresis include; Separation Detection Quantification
  • 27. a. Electrophoresis Separation DrAnurag yadav,Bio-FMMC27  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 DrAnurag yadav,Bio-FMMC28  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 DrAnurag yadav,Bio-FMMC31  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.
  • 33. What is EEO & why low???
  • 38. Common effect of variables on separation DrAnurag yadav,Bio-FMMC38 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 39
  • 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
  • 48. References DrAnurag yadav,Bio-FMMC48  KeithWilson- Principles and techniques of biochemistry and molecular biology.  Upadhyay- biophysical chemistry.  Tietz-Text book of clinical chemistry.  Kaplan- clinical chemistry.  YouTube and Google images.