This presentation contain the information about gel electrophoresis method , instruments & types. Electrophoresis is a method through biological molecules are separated by applying an electric field. Main purpose of this method is to determine the number , amount & mobility of biological component. There are some internal & external factors that affects the process of electrophoresis. The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated There are 3 types of gels that use in this process . In this buffers are also used which provide ions that carry a current.

1. Electrophoresis

2. Definition
• Electrophoresis is a method whereby charged molecules in solution,
chiefly proteins and nucleic acids, migrate in response to an electrical
field.
• This technique was firstly developed by the Arne Tiselius in 1930 for the
study of serum protein.
• Electrophoresis is a technique used in laboratories in order to separate
macromolecules based on size.

3. Purpose of Electrophoresis:
• To determine the number, amount & mobility of components in a given
sample or to separate them.
• To obtain the Information about the electrical double layer surrounding
the particles.
• To determine the molecular weight of protein molecules.

4. Factors affecting Electrophoresis
Inherent Factors:
• Magnitude of its charge
• Charge density
• Molecular weight
External Environmental
Factors:
• Solution Ph
• Electric Field
• Solution Viscosity
• Temperature

5. Electrophoretic
Chamber

7. Principle of Electrophoresis
• Positive & negative electrical charges are frequently associated with
biomolecules.
• Electrophoresis of positively charged particles (cations) is called
cataphoresis, while electrophoresis of negatively charged particles
(anions) is called anaphoresis.
• When they placed in electric field , charged biomolecules move towards
the electrodes of opposite charge due to the phenomenon of electrostatic
attraction.
• An ampholyte become positively charged in acidic conditions & migrate to
cathode, In alkaline conditions they become negatively charge & migrate
to anode.

8. • Can be represented by the following equation:
u=v/E = q/f
v = velocity of migration of molecules
E = Electric Field in volts per cm
q = Net electric charge on the molecule
f = frictional coefficient
• Electrophoretic mobility of the molecules is directly proportional to charge
density.
• Higher the charge greater the electrophoretic mobility.

9. Types of Gels:
• There are 3 types of gels that are use in electrophoresis:
1. Agarose:
For separating larger nucleic acids
2. Polyacrylamide gel:
For separating smaller nucleic acids.
3. SDS-PAGE:
For denaturing the proteins.

11. Buffers:
• Buffers in gel electrophoresis are used to provide ions that carry a
current & maintain the pH at a relatively constant value.
• The buffer ionic strength will determine the thickness of the ionic cloud.
• Buffers used are made monovalent ions because their valences & molality
are equal.

13. Technique:
1. Sampling
2. Electrophoretic run
3. Staining
4. Detection & Quantification

14. 1- Sampling
• The sample is allow to fall into the sample wells.
• The sample may applied as a spot about 0.5cm in diameter or as uniform
streak.
• The sample is then placed into the electrophoretic chamber in contact
with the buffer.

15. 2- Electrophoretic run
• The current is switched on after the sample has been applied to the paper
& the paper has been equilibrated with the buffer.
• The types of buffer used depends upon the type of separation.
• It will cause the negatively charged proteins or nucleic acids to migrate
across the gel away from the negative electrode.
• Smaller biomolecules travel farther down the gel, while lager ones remain
closer to the point of origin.

16. 3- Staining
• The sample is then stained & dried after washing out the excess dye.
• Amido Black B or members of Coomassie Brillant Blue series are the
commonest dyes.
• The amount of dye taken up is dependent on the type of protein, degree of
denaturation & quality of dye.

17. 4- Detection & Quantification:
• Detection can be achieved by using UV light.
• DNA may be visualized using ethidium bromide which then intercalated
into DNA, fluorescence under UV light
• Proteins may be visualized using silver stain or Coomassie Brillant Blue
dye.

19. Downstream Processing
• After separation, An additional separation method may be used.
• The gel will then be physically cut & the protein complexes extracted from
each portion separately.
• This can provide a great deal of information about the identities of the
proteins in a complex.

20. Types of Electrophoresis
1. Zone Electrophoresis
2. Slab gel Electrophoresis
3. Disc Electrophoresis
4. Isoelectric Focusing Electrophoresis
5. 2 Dimensional Electrophoresis
6. Capillary Electrophoresis

21. 1- Zone Electrophoresis
• Produce zone of proteins that are heterogeneous & physically separated
from one another.
• Classified according to type & structure of the support material e.g. AGE,
CAE, PAGE etc.

22. 2- 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 serum proteins, isoenzymes, lipoproteins,
hemoglobin & fragments of DNA & RNA.

24. 3- Disc Electrophoresis
• 3 Gel system
1- Small pores separating gel (running gel)
2- Larger pore separating gel (stacking gel)
3- Thin layer of large pore monomer solution (sample gel)
• All proteins migrate easily through the large-pore gels.
• This improves resolution & concentrate protein components at the border.
• Discontinuities in electrophoretic matrix caused by layers of gels.

26. 4- Isoelectric Focusing Electrophoresis
• It is separation method that resolves proteins markers on the basis of
their isoelectric points.
• Proteins migrate through a zone in a medium where the pH of the gel
matches its PI.
• At this point, the charge of the protein becomes zero & its migration
ceases. It becomes focused.
• A high voltage power source is needed because carrier ampholytes are
used in relatively high concentrations. Thus it must be cooled.
• It is to test for variant Hb. Also use in clinical laboratories for muscle
extract & serum extract.

28. 5- 2-Dimensional Electrophoresis
• This technique combines the techniques of IEF which separates proteins
in a mixture according to charge (PI) with the size separation technique of
SDS-PAGE.
• 1st Dimension – Charge dependent IEP
• 2nd Dimension – Molecular weight dependent electrophoresis
• It achieves the highest resolving power for the separation of DNA
fragments.

30. 6- Capillary Electrophoresis
• Separation in narrow bore fused silica capillaries filled with buffer.
• Sample is loaded after filling capillary with buffer & electric field applied.
• Electro-osmotic flow (EOF) controls the amount of time solute remain in
the capillary.
• Cations migrate fastest due to EOF & electrophoretic attraction towards
the cathodes.
• Anions move slower because EOF is slightly greater than the attraction
towards the anode & repulsion from cathode.

32. Applications
• DNA sequences can be isolated, analyzed & cloned.
• Synthesis of new antibiotics.
• Analysis of bacteria in response of antibiotics.
• Purifications, processing, & analysis of vaccines e.g. polio vaccine.
• Protein & DNA analysis.
• Determination of impurities.
• Analysis of carbohydrates & macromolecules.
• Analysis of inorganic anions/ metal ions.

33. • Molecular biology, Microbiology, Biochemistry.
• Use in DNA fingerprinting.
• Separation of serum proteins.
• Use in antigen-antibody species.
• Use in food industry