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General principles of electrophoresis

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general principles & factors affecting electrophoresis .;a practical approach

general principles & factors affecting electrophoresis .;a practical approach

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  • 1. General Principles of Electrophoresis Dr. Gangadhar Chatterjee 23/07/2014
  • 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. 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. 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. Where are the charges from?  Proteins
  • 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. 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.  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. high OH-High H+
  • 10. What can we deduce from it?
  • 11. Factors Affecting Electrophoresis
  • 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.  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. 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. Effect of matrix concentration
  • 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. Electrophoresis Equipment: Horizontal or Submarine Gel DNA/RNA is negatively charged: RUN TO RED
  • 18. Agarose Gel Electrophoresis System
  • 19. Homogeneous buffer Discontinuous buffer
  • 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. Types of Electrophoresis  Agarose, cellulose, polyacrylamide  Iso-electric focusing  Capillary electrophoresis  Two-dimensional electrophoresis
  • 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. Destaining solution used in most cases are Methanol and acetic acid mixture only.
  • 24. Few technical considerations
  • 25. What is EEO & why low???
  • 26. Electroendosmosis cont
  • 27. Normal patterns Plasma protein Hemoglobin Alkaline pH DNA Normal
  • 28. Troubleshooting
  • 29. Thanks