Electrophoresis is the migration of charged particles through an electric field towards the electrode of opposite charge. It works by separating molecules based on their size and charge. Key factors that affect electrophoresis include the net charge and size/shape of molecules, as well as the ionic strength, pH, temperature, and type of support medium used. Common applications include separating proteins, nucleic acids, and other biological molecules to analyze samples.
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
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
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
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.
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.
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
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
Immunoelectrophoresis Agar gel slab Separation of
antigenic proteins