It describes migration of charged particles or molecules under the influence of electric field.
PURPOSE FOR CARRYING OUT ELECTROPHORESIS :-
1. To determine the number, amount and mobility of components in a given sample or to separate them.
2. To obtain information about the electrical double layers surrounding the particles.
3. Determination of molecular weight of proteins and DNA sequencing.
Any charged ion or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge, size, shape and the applied electric current.
Can be represented by following eq.
• v = velocity of migration of the molecule.
• E = electric field in volts per cm
• q = net electric charge on the molecule
• f = frictional coefficient
The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility ,denoted by
For molecules with similar conformation f varies with size but not with shape. Thus electrophoretic mobility (p) of a molecule is directly proportional to charge density(charge\mass ratio).
FACTORS AFFECTING ELECTROPHORETIC MOBILTY :-
1. Charge — higher the charge greater the electrophoretic mobility.
2. Size — bigger the molecule greater are the frictional and electrostatic forces exerted on it by the medium. Consequently, larger particles have smaller electrophoretic mobility compared to smaller particles.
3. Shape — rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours. Therefore globular protein move faster than fibrous protein.
TYPES OF ELECTROPHORESIS:-
- FREE ELECTROPHORESIS :- a. Micro electrophoresis and b. Moving boundary
- ZONE ELECTROPHORESIS:- a. Paper electrophoresis,b. Cellulose active and c. Gel electrophoresis
It is the form of electrophoresis that is carried out on filter paper. This technique is useful for separation of small charged molecules such as amino acids and small proteins.
FILTER PAPER :- It is the stabilizing medium. We can use whatman filter paper,cellulose acetate filter paper or chromatography paper.
APPARATUS :- Power pack, electrophoretic cell that contains electrodes, buffer reservoirs, support for paper, transparent insulating cover.
SAMPLE APPLICATION :- The sample may be applied as a spot(about 0.5 cm in diameter)or as a uniform streak.
ELECTROPHORETIC RUN:-The current is switched on after the sample has been applied to the paper and the paper has been equilibrated with the buffer.The types of buffer used depends upon the type of separation.Once removed. the paper is dried in vaccum oven.
DETECTION AND QUANTITATIVE ASSAY:-
To identify unknown components in the resolved mixture the electrophoretogram may be compared with another electrophoretogram on which standard components have been electrophoresced under identical conditions.Physical properties like fluorescence,ultraviolet absorption or radioactivity are exploited for detection.
GEL ELECTROPHORESIS :-
It is a technique used for the separation of Deoxyribonucleic acid. Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix.
• what is a gel?
Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules.
Types of Gel:
• Agarose gel.
• Polyacrylamide gel.
AGAROSE GEL :-
• A highly purified uncharged polysaccharide derived from agar.
• Used to separate macromolecules such as nucleic acids, large proteins and protein complexes.
• It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C.
• It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds.
POLYACRYLAM IDE GEL
• Commonly used components: Acrylamide monomers, Ammonium persulphate, Tetramethylenediamine (TEMED), N.N’-methylenebisacrylamide.
• These free radicals activate acrylamide monomers inducing them to react with other acrylamide monomers forming long chains.
Used to separate most proteins and small oligonucleotides because of the presence of small pores.
ELECTROPHORESIS OF PROTEINS
The most commonly used technique for the separation of proteins is Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE).
Protein sample is first boiled for 5 mins in a buffer solution containing SDS and 3-mercaptoethanol. Protein gets denatured and opens up into rod-shaped structure.
Sample buffer contains bromophenol blue which is used as a tracking dye, and sucrose or glycerol. Before the sample is loaded into the main separating gel a stacking gel is poured on top of the separating gel. Current is switched on. The negatively charged protein-SDS complexes now continue to move towards the anode. As they pass through the separating gel, the proteins separate, owing to the molecular sieving properties of the gel. when the dye reaches the bottom of the gel, the current is turned off. Gel is removed from between the glass plates and shaken in an appropriate stain solution. Blue colored bands are observed under UV rays.
CONTINUOUS AND DISCONTINUOUS BUFFER
A continuous buffer system has only a single separating gel and uses the same buffer in the tanks and the gel.
In a discontinuous system a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel.
E.g. SDS PAGE Electrophoresis.
• The resolution obtainable in a discontinuous system is much greater than that obtainable in a continuous one. However the continuous system is easier to set up.
1.Which factors are affecting electrophoresis mobilty ?
b. Shape of protine
c. a and b
d. none of the above
2. Which is not a type of zone electrophoresis ?
a. Gel electrophoresis
c.Cellulose active electrophoresis
d. Micro electrophoresis
3. Which technique separates charged particles using electric field?
c. Protein synthesis
d. Protein denaturing
4. Which of the following statements is true about migration of biomolecules?
a. The rate of migration is directly proportional to the resistance of medium
b. Rate of migration is directly proportional to current
c. Low voltage is used for separation of high mass molecules
d. Rate of migration is inversely proportional to current
5. What does the electrophoresis apparatus consist of?
a. Gel, buffer chamber and fire pack
b. Buffer chamber and electrophoresis unit
c. Electrophoresis unit and gel separator
d. Power pack and electrophoresis unit
6. Which type of paper are used in paper electrophoresis?
a.whatman filter paper
b.cellulose acetate filter paper
d. All of the above
7. what is agarose gel ?
a. Cross linkage molecules
b. Purified unchange poly saccharide
c.It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C
d. All of the above
Text book of pharmaceutical analysis third edition by Dr.S.Ravi sankar (Pg no. 28.1-28.8)