electrophoresis: movement of charge particles in a gel under the influence of an electric field, principle, factors, apparatus, types , application, advantage and disadvantage.

1. ELECTROPHORESIS
Mohd Kaif Khan
B.sc final year (botany department)
Aligarh Muslim university AMU Aligarh,
202002

2. (father of electrophoreses)
Arne Tiselius

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.

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.

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

23. References
• Tietz-textbook of clinical chemistry
• Kaplan-clinical chemistry.
• Youtube and Google images.

24. THANK
YOU