General Principles of Electrophoresis
Dr. Gangadhar Chatterjee
23/07/2014
Electrophoresis
 a separation technique
 Simple, rapid and highly sensitive
 used in clinical laboratories to separate ...
Clinical applications of Electrophoresis
 Serum Protein Electrophoresis
 Lipoprotein Analysis
 Diagnosis of Haemoglobin...
Typical oligoclonal bands
found only in CSF in
Multiple sclerosis ( IEF &
Western blotting)
Diagnosis of CJD by Immunoblot...
Where are the charges from?
 Proteins
General Principles
 An idealized, simplified situation: an isolated charged
particle in a non conducting medium.
 The fo...
Electrophoretic mobility U (the ratio of velocity to the
strength of the driving field)
U = v/E = Ze/f
The zonal techniq...
 Almost all electrophoresis of biological macromolecules
is at present carried out on either polyacrylamide or
agarose ge...
high OH-High H+
What can we deduce from it?
Factors Affecting Electrophoresis
Interrelation of Resistance, Voltage, Current
and Power
 Two basic electrical equations are important in
electrophoresis
...
 Under constant current conditions (velocity is directly
proportional to current), the velocity of the molecules is
maint...
Temperature and Electrophoresis
Important at every stage of electrophoresis
 During Polymerization
- Exothermic Reaction
...
Effect of matrix concentration
Agarose (%)
Range of separation of linear DNA
(in kilobases)
0.3 60 - 5
0.6 20 - 1
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1....
Electrophoresis Equipment: Horizontal or
Submarine Gel
DNA/RNA is negatively charged: RUN TO RED
Agarose Gel Electrophoresis System
Homogeneous
buffer
Discontinuous
buffer
Continuous and Discontinuous Buffer
Systems
 A continuous system has only a single separating gel
and uses the same buffe...
Types of Electrophoresis
 Agarose, cellulose, polyacrylamide
 Iso-electric focusing
 Capillary electrophoresis
 Two-di...
Different stains of Electrophoresis
 Plasma Proteins
- Amido black
- Coomassie Brilliant Blue
- Bromophenol Blue
 Hemogl...
Destaining solution used in most cases are Methanol and acetic acid mixture only.
Few technical considerations
What is EEO & why
low???
Electroendosmosis cont
Normal patterns
Plasma protein Hemoglobin
Alkaline pH
DNA
Normal
Troubleshooting
Thanks
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
General principles of electrophoresis
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General principles of electrophoresis

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general principles & factors affecting electrophoresis .;a practical approach

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General principles of electrophoresis

  1. 1. General Principles of Electrophoresis Dr. Gangadhar Chatterjee 23/07/2014
  2. 2. 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
  3. 3. 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)
  4. 4. Typical oligoclonal bands found only in CSF in Multiple sclerosis ( IEF & Western blotting) Diagnosis of CJD by Immunoblot after SDS- PAGE separation of A) Human brain protein B) CSF of CJD patient C) Normal CSF
  5. 5. Where are the charges from?  Proteins
  6. 6. General Principles  An idealized, simplified situation: an isolated charged particle in a non conducting medium.  The force experienced by a particle in an electrical field is given by Coulomb’s law,  F = ZeE (E-electric field: potential per unit length)  The viscous resistance of the medium to the motion: -fv (f: the frictional factor)  The viscous resistance of the medium just balances the driving force. fv = F = ZeE
  7. 7. Electrophoretic mobility U (the ratio of velocity to the strength of the driving field) U = v/E = Ze/f The zonal techniques: In these methods, a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix. The kind of supporting matrix used depends on the type of molecules to be separated and on the desired basis for separation: charge, molecular weight, or both.
  8. 8.  Almost all electrophoresis of biological macromolecules is at present carried out on either polyacrylamide or agarose gels  The matrix provides stability against convection and diffusion. In addition, in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size.  allows a permanent record of results through staining after run
  9. 9. high OH-High H+
  10. 10. What can we deduce from it?
  11. 11. Factors Affecting Electrophoresis
  12. 12. Interrelation of Resistance, Voltage, Current and Power  Two basic electrical equations are important in electrophoresis  The first is Ohm's Law, I = V/R  The second is H = VI ( heat produced per unit time)  This can also be expressed as H = I2R  In electrophoresis, one electrical parameter, either current, voltage, or power, is always held constant Ideally voltage is kept constant. WHY?
  13. 13.  Under constant current conditions (velocity is directly proportional to current), the velocity of the molecules is maintained, but heat is generated.  Under constant voltage conditions, the velocity slows, but no additional heat is generated during the course of the run  Under constant power conditions, the velocity slows but heating is kept constant
  14. 14. Temperature and Electrophoresis Important at every stage of electrophoresis  During Polymerization - Exothermic Reaction -Gel irregularities -Pore size  During Electrophoresis -Denaturation of proteins -Smile effect -Temperature Regulation of Buffers
  15. 15. Effect of matrix concentration
  16. 16. Agarose (%) Range of separation of linear DNA (in kilobases) 0.3 60 - 5 0.6 20 - 1 0.7 10 - 0.8 0.9 7 - 0.5 1.2 6 - 0.4 1.5 4 - 0.2 2.0 3 - 0.1
  17. 17. Electrophoresis Equipment: Horizontal or Submarine Gel DNA/RNA is negatively charged: RUN TO RED
  18. 18. Agarose Gel Electrophoresis System
  19. 19. Homogeneous buffer Discontinuous buffer
  20. 20. Continuous and Discontinuous Buffer Systems  A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel  In a discontinuous system a nonrestrictive large pore gel, called a stacking gel, is layered on top of a separating gel  The resolution obtainable in a discontinuous system is much greater than that obtainable in a continuous one. However, the continuous system is a little easier to set up.
  21. 21. Types of Electrophoresis  Agarose, cellulose, polyacrylamide  Iso-electric focusing  Capillary electrophoresis  Two-dimensional electrophoresis
  22. 22. 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
  23. 23. Destaining solution used in most cases are Methanol and acetic acid mixture only.
  24. 24. Few technical considerations
  25. 25. What is EEO & why low???
  26. 26. Electroendosmosis cont
  27. 27. Normal patterns Plasma protein Hemoglobin Alkaline pH DNA Normal
  28. 28. Troubleshooting
  29. 29. Thanks
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