Electrophoresis

2. Electrophoresis is the migration of
charged particle in an electric field
towards the electrode bearing the
opposite charge.

3. PRINCIPLE
Many important biological molecules such as
aminoacids,peptides,proteins,nucleotides &
nucleic acid posses ionizable groups & therefore at
any given pH exist in solution as electrically
charged species, either as anions (-) or cations
(+).Under the influence of an electrical field these
charged particles (cations) migrate to the cathode
(Negative electrode) or (anions) move to the
anode( positive electrode), depending on the
nature of their net charge.

4. FACTORS AFFECTING ELECTROPHORESIS
1.Net charge on the molecule
Grater charge grater mobility
2.Size & shape of molecule
Grater size ↓se mobility
Shape Globular -----------↑se mobility
Fibrous ----------- ↓se mobility

5. 3.Ionic strength & pH of buffer
↑ se ionic strength ↑se heat production ↓se mobility
↓se ionic strength less heat production ↑se mobility
pH of buffer 1-11
4.Strength of electric field
5.Temprature
6.Nature of support medium

6. TYPES OF ELECTROPHORESIS

7. ELECTROPHORESIS APPARATUS
 Electrophoresis tank –
Buffer chamber
Electrodes
Electrical connection to power
supply
Space for keeping support
medium
 Power tank – to provide constant currents or
constant voltage

8. ELECTROPHORETIC TANK AND POWER PACK

9. APPARATUS
1.Electrophoresis is carried out in a tank suitable for
supporting medium e.g. paper or gel.
Major part of tank include –
Buffer chamber
Electrodes
Electrical connection to power supply
Space for keeping support medium
i.e. paper & slides of agar gel.
2.Power pack

10. SUPPORT MEDIUM
Filter paper
Cellulose acetate membrane
Agar & agarose
Starch
Polyacrylamide

11. ELECTROPHORETIC TANK AND POWER PACK

12. ADDITIONAL REQUIREMENTS
Buffer
Fixative
Staining solution
Destaining solution
Densitometer – is essentially a double beam filter
photometer or spectrophotometer that scans the
electrophoretic strip ( in the form of agarose ,
cellulose acetate or polyacrylamide) as it moves
past the optical system.

13. DENSITOMETER

14. PROCEDURE
1. Sample to be separated is applied to a supporting medium
(paper, cellulose acetate, agar gel, polyacrylamide gel etc.)
2. Electrophoresis is carried out at desired constant voltage
or constant current in presence of specific pH.
3. After completion of electrophoresis the supporting
medium is placed in a fixative to prevent diffusion of
separated fractions.
4. Separated fraction is then visualized by using appropriate
stains e.g. Bromophenol Blue & Amino Schwartz for
plasma proteins & Sudan Black for lipoproteins.
5. Quantification of each fraction is done by either
densitometer or elution followed by colorimeter or
spectrophotometer of eluted fraction.

15. RUNNING OF ELECTROPHORESIS

16. APPLICATIONS
1.Separation of
biological molecules
like plasma proteins ,
nucleic acids,
nucleotides, charged
carbohydrate
derivatives,
glycoproteins,
lipoproteins,
hemoglobin variants,
isoenzymes etc.

17. 2.Analytical Applications-
Determination of DNA sequences
Southern & Northern Blotting
Restriction Mapping of DNA
3. In Protein Study
Determination of subunit stoichiometry
Determination of molecular weight

18. TECHNIQUE
APPICATION OF SAMPLE
RUNNING OF SAMPLE
VISUALISATION OF SAMPLE
QUANTIFICATION

19. APPLICATIONS
Types of
electrophoresis
Support medium applications
paper Whatman’s paper no.1 Separation of
peptides
Plasma proteins
Nucleic acid
Charged carbohydrates
Cellulose acetate Cellulose acetate strips
in which hydroxyl gr.of
cellulose is acetate
with acetic anhydride
Separation of
Glycoproteins
Lipoproteins
Hb derivatives
Gel :- Based on charge & molecular size
a. Agarose gel Composed of agarose &
agaropectin
Separation of
plasma proteins
Lipoproteins
Hb derivatives

20. CONTINUED
b. Starch Partially hydrolysed
starch
Separation of
isoenzymes
c. polyacrylamide Polyacrylamide gel
layers of varying pore
size
Separation of
Glycoproteins
Lipoproteins
Hb derivatives
DNA fragments
Isoelectricfocussing Polyacrylamide gel Separation of
plasma proteins
Lipoproteins
Hb derivatives
ImmunoelectrophoresisAgar gel slab Separation of
antigenic proteins

21. TYPES OF PLASMA PROTEINS
Albumin
Globulins-
α
1
, α
2,
β,γ globulins

23. How To write in journal
Defination
Factors affecting electrophoresis
Types of electrophoresis
Procedure
Applications