electrophoresis: movement of charge particles in a gel under the influence of an electric field, principle, factors, apparatus, types , application, advantage and disadvantage.
3. CONTANT
• Introduction.
• Principle.
• Factors affecting.
• Electrophoresis apparatus.
• General operation.
• Technical and practical consideration.
• Types of electrophoresis.
4. ELECTROPHORESIS
• “Electro” means “charged particle” and “phoresis” means
“movement", it simple describes the migration pf a charged particle
under the influence of electric field.
• Electrophoresis is the migration of charged particles or molecules in a
medium under the influence of an applied electric field.
• Many important biological molecule such as a amino acids, peptides,
proteins, nucleotides, nucleic acids posses ionizable groups and
therefore, at any given pH, exists in solution as electrically charged
species either as cations or anions.
• This is one of the most fundamental processes used in all types of
molecular biology and RDT experiments
5. PRINCIPLE
• Under the charge of an electric field these charged particles will
migrate either to cathode or to anode, depending on the nature of
their net charge.
• This is one of the most fundamental processes used in all types of
molecular biology and RDT experiments
6. • In the process of electrophoresis large molecule have more difficulty
in moving through the supporting medium (i.e., gel) where as the
smaller medium has more mobility through it.
7.
8. Electrophoretic mobility
• Electrophoretic mobility is defined as the rate of migration (cm/sec)
per unit field strength (volts/cm).
• µ=Q/6πr η
• Where,
µ=electrophoretic mobility
Q= net charge on the ion
r=Ionic radius of the solute
η=Viscosity of the medium
9. Factors effecting
• The rate of migration of charged molecules depends upon following
factors;
• 1) the strength of electric field, size and shape.
• 2) relative hydrophobicity of the sample.
• 3) ionic strength and temperature of the buffer.
• 4) molecular size of the taken bio molecule.
• 5)net charge density of the taken bio molecule.
• 6) shape of the taken bio molecule
10. • The different components in a mixture will have different
electrophoretic mobilities and hence they can be separataed.
• Mixtures of amino acids, proteins and nucleotides can be separated
by their migration in an electric field.
11. Electrophoresis apparatus
• Apparatus includes-
• 1)buffer solution
• 2)buffer tank-to hold the buffer
• 3)electrodes-made of platinum or carbon
• 4)power supply
• 5)support media
• Note-Choice of buffer depends on the nature of substance to be
separated and the electricity is supplied at a constant current and
voltage.
12.
13. • Buffer
• Its carry applied electric current.
• They set the pH at which electrophoresis is carried out.
• Supporting medium
• It is a matrix in which protein separations takes place.
• Separation based on charge and mass of the biomolecule and pore
size of the medium.
14. Types of electrophoresis
• A)Zone electrophoresis
• 1)Paper electrophoresis
• 2)Gel electrophoresis
• 3)Isoelectric focusing
• 4)Agarose gel
• B) Moving boundary electrophoresis
• 1)Capillary electrophoresis
• 2)Isoelectrophoresis
15. Gel electrophoresis
• Separation is brought about through molecular sieving technique,
based on the molecular size. Gel material acts as a “molecular sieve”.
• Gel is a colloid in a solid form (99% is water).
• Different types of gels which can be used as are; Agar, Agrose gel ,
Starch, Polyacrylamide gels.
16. Agarose gel
• 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.
• Used to separate DNA, Proteins, Hb variants, iso-enzymes etc.
17. ISOELECTRIC FOCUSING
• Separation of proteins according to the iso-electric points
• It is ideal for separation of amphoteric substances.
• Separation is achieved by applying a potential difference across a gel
that contain a pH gradient
• Isoelectric focusing requires solid support such as agarose gel and
polyacrylamide gel.
• It gives good separation with a high resolution compared to any other
methods.
18. POLYACRYLAMIDE GEL ELECTROPHORESIS
• Frequently referred as PAGE.
• Cross-linked polyacrylamide gel are formed from the polymerization
of the monomer in presence of small amount of N,N”-methylene-
bisacrylamide.
• Two acrylamide linked by the methylene group.
• It is an example for free radical catalysis.
• Low % has large pore size and vice verse.
19. SDS-PAGE
• Sodium dodecyle sulphate-polyacrylamide get electrophoresis.
• Most widely used method for analysing protein mixture qualitively.
• Useful for monitoring protein purification –as separation of protein is
based on the size of the particle.
• Ca also be used for determining the relative molecular mass of the
protein.
• Marcaptoethenol will break the disulphide bridges.
• SDS binds strongly to and denatures the protein in its native shape
and side
20. APPLICATION
• Research tool
-measuring molecular weight
-peptide mapping
-protein identification
-determination of sample purity
• Separation of proteins and establishing size
• Smaller fragments of DNA
21. • ADVANTAGES:
Gels are stable over wide range of pH and temperatures.
Gels of different pore size can be formed
Simple and separation speed is good comparatively.
Exhibit resonabale mechanical strength.
• DISADVANTAGES:
GEL preparation and casting is time-consuming.
Carcinogenic
Complete reproducibility of gel preparation not possible.
22. • Staining
• Fluresenct stains-ethidium bromide-nucleic acids
• Silverstain for protein gel
• Dye based –Coomassie blue-protein band.
• Tracking dyes-BPB>xylene cyanol
