Download - General principles of electrophoresis
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General Principles of Electrophoresis
Dr. Gangadhar Chatterjee23/07/2014
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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
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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)
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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
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Where are the charges from?
Proteins
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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
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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.
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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
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high OH-High H+
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What can we deduce from it?
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Factors Affecting Electrophoresis
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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?
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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
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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
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Effect of matrix concentration
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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
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Electrophoresis Equipment: Horizontal or Submarine Gel
DNA/RNA is negatively charged: RUN TO RED
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Agarose Gel Electrophoresis System
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Homogeneous buffer
Discontinuous buffer
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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.
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Types of Electrophoresis
Agarose, cellulose, polyacrylamide
Iso-electric focusing
Capillary electrophoresis
Two-dimensional electrophoresis
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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
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Destaining solution used in most cases are Methanol and acetic acid mixture only.
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Few technical considerations
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What is EEO & why low???
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Electroendosmosis cont
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Normal patterns
Plasma protein HemoglobinAlkaline pH
DNANormal
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Troubleshooting
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Thanks