Intriduction about Slab gel Electrophoresis in Bio Instrumetion Protein separation method Electrophoresis method

1. Slab Gel
Electrophoresi
s
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2. Content
 Introduction
 Principle of Electrophoresis
 Factors affecting Electrophoretic mobility
 Conventional Electrophoresis
 Slab Gel Electrophoresis
 Application Of Electrophoresis
 Advantages & Disadvantages
 References
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3.  A separation technique
 Simple, rapid and highly sensitive
 used in clinical laboratories to separate charged
molecules from each other in presence of electric
field
 Like
– Proteins in bodyfluids: serum, urine,CSF
– Proteins in erythrocytes: haemoglobin
– Nucleic acids: DNA,RNA
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4. Principle Of Electrophoresis
Electrophoresis is the migration
of charged particle or molecule
in the medium under the
influence of an applied electric
field.
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5.  Depending on kind of charge the molecule carry,
they move towards either
 Tocathode
 Or to Anode
 Anampholytes become positively charged in acidic
condition and migrate to cathode, in alkaline
condition they become negatively charge and
migrate to anode.
 Ampholytes are amphoteric molecules that contain
both acidic and basic groups and will exist mostly
as zwitterions in a certain range of pH.
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6.  The pH at which the average charge is zero is
known as the molecule's isoelectric point.
Ampholytes are used to establish a stable pH
gradient for use in isoelectric focusing.
 Eg: asprotein contain the ionisable amino and carboxyl
group.
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7. Factors affecting
Electrophoretic mobility
 The rate of migration of an ion in electrical field
depend onfactors,
1. Net charge of molecule
2. Sizeand shape of particle
3. Strength of electrical field
4. Properties of supporting
medium
5. Temperature of operation
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8. Mobility
Under the electrical field, the
mobility of the particle is
determined by two factors:
 Its charge
 Frictional coefficient
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9.  Sizeand shape of the particle decide the velocity
with which the particle will migrate under the given
electrical field and the medium
 Where is Small ,Round & Highly Charged
 is Large , Star & Less charged
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10. Strength of Electricalfield
 The rate of migration under unit potential
gradient is referred as mobility of ion.
 Greater the potential gradient greater the
migration
 Also increase in potential difference leads to
rising of system temperature results in
decreases the viscosity of medium
consequently increases the movement of ions.
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Figure: Strength Of Electrical field

12.  As the molecule exist asamphoteric ,they will carry the charges
basedon the solvent pH.
 Their overallnet charge isNEUTRALwhen it isat zwitter ion
state. Andhence the mobility isretarded to zero.
 Mobility isdirectly proportional to the magnitudeof the
charge, whichisfunctional onthe pH of solvent.
 The pH ismaintained bythe use ofBuffersofdifferent pH.
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13. overview
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Instrumentation :
 Tworeservoir for thebuffer
 Powersupplyand Electrodes
 Separation medium
Conventional Electrophoresis

15. Buffer
 The buffer in electrophoresis hastwo fold purpose:
 Carry applied electrical current
 Theyset the pH aswhich electrophoresis iscarried out.
 Thus they determine;
 Typeof 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.
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16. Commonly Used Buffers
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17. 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) ImmunoElectrophoresis
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18. • Traditional methods, using a
rectangular gel regardless of
thickness
• DISContinuities in
electrophoretic matrix causedby
layers of
polyacrylamide/starch gelt
hat differ in composition &pore size
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CLASSIFICATION
Slab Gel
Electrophoresis
Disc
Electrophoresis

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Slab Gel Electrophoresis
 It is primary method used in
Clinical chemistry lab.
 It has ability to
Simultaneously separate
Several samples in one run.
 It uses a rectangular gel
Regardless of thickness.
 Gels are cast on sheets of
Plastic backing.
 It is useful in separation of isoenzymes,
Lipoproteins, Hemoglobin & fragments
Of DNA & RAN

20. Slab Gel Electrophoresis is a type
of Gel electrophoresis
The gel is set or polymerized into
a thin slab between two glass
plates.
The thickness of the slab of the
gel can be adjusted by placing
spacers of various thickness
between the two glass plates.
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21. Slab gel electrophoresis (SGE)
is an established technique for
protein separation,
whereby many samples can be
analysed simultaneously in a 1D
format or an extremely complex
mixture of proteins can be
resolved in a 2D format.
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22. Preparation Of Cast For
Slab Gel
Sample wells are made at one end of
the gel by placing a comb-
shaped jig into the gel before it sets or
polymerizes.
After the gel has set, the comb is
removed leaving the sample wells
etched into the gel.
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Figure: Casting Of Slab gel

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25. Since a number of wells can be cast
side-by-side, number of samples can
be loaded simultaneously and
compared under conditions which
remain essentially identical.
This is a great advantage of this
technique.
This technique is become extremely
popular, especially in the field of
molecular biology.
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26. Slab Gel Units
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27. Application
1) DNA Sequencing
2) Medical Research
3) Protein research/purification
4) Separation of organic acids, alkaloids,
carbohydrates, amino acids, alcohols, nucleic
acid etc…
5) In food industry
6) For testing purity of thyroid hormones
7) Quantitative separation of all fractions of
antibiotics, RBCs and Enzymes
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28. Advantages &
Disadvantages
 Advantages
 Easy to prepare and small concentration of
gel is required
 Resolution is superior to that of filter paper.
 Large quantities of proteins can be separated
and recovered.
 It adsorbs proteins relatively less when
compared to other medium
 Recovery of protein is good, good method for
preparative purpose.
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29. Advantages &
Disadvantages
 Disadvantages
 Electro osmosis is high.
 Resolution is less compared to other
methods.
 Different sources and batches of agar tend to
give different results and purification is often
necessary.
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30. References
 Keith Wilson- Principles and techniques of
biochemistry and molecular biology.
 “Biophysical chemistry –principle and
technique by Upadhyay-Upadhyay-Nath
 Advance in protein chemistry 65th edition
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