What is ELECTROPHORESIS TECHNIQUE
The movement of charged particles under the influence of an electric current to oppositely charged electrode is known as Electrophoresis" An analytical technique where particle charge ion and can be separated from a mixture sometimes known as Ionophoresis. Ex: Seperation of Ions.
"The movement of charged particles under the influence of an electric current to oppositely charged electrode is known as Electrophoresis"
An analytical technique where particle charge ion and can be separated from a mixture sometimes known as Ionophoresis. Ex: Seperation of Ions.
Since protein exists as charged particles Electrophoresis method is widely used for the separation of proteins in biological fluids. The term Electrophoresis first used by Arne Tiselius in 1937 was awarded Nobel Prize in 1940. The process of electrophoresis was examined in radiation of colloidal particles which were charged and can more in electrical field either cathode or anode depending upon charge they have.
A technique by which molecules like enzyme, Proteins, Amino acids. Nucleotide and nucleic Acid are separated by difference in the net charge in the presence of external applied electrical fields.
Principle of Technique
: According to theoretical condition separation of charged particle it done by passing an electrical current.
"Cat ions moves towards cathode and Anions moves towards Anode"
The distance they more depends upon certain factors
• Time of Migration
• Electrical Charge
• Size of Solvent Particles
The variable controls the movement of charged particle under electrical field and conductivity of solvent can be kept constant as well as time and amount of current can also be kept constant and particles are separated on basis of their charge and size in electrophoresis equipment.
: Movement of charged particle the distance travelled by charged particle in one second then a current of 1 volt /cm is applied.
The mobility can be represented with a symbol
µ = V/E
V= Migration Velocity (Distance Travelled in cm ), E = Electric Current (Volt/cm)
Since particle of different size have different electrophoretic mobility induce in the influence of some current. It is possible to separate them from mixture.
Ohm's Law for Relationship Between Electrical Parameters: The Law which describes the relationship between the voltage 'V' the current 'I' and resistance 'R' in a DC circuit. A greater voltage produces greater current by given resister:
V = IR
Variation of Ohms rule describes a small changes in electrical current can produce large changes in power expenditure (P).
P = IV = I 2 R
This technique is routinely used for enzyme purification and isoenzyme separation in the laboratory. All through it has found only limited application at large scale. Since the technique is time consume and which expensive. Various type of instrumental approach has use to separate and purified charge molecules using electrophoresis.
Basically there are two type of electrophoresis, Free Electrophoresis and Zone Electrophoresis
Frontal Electrophoresis or Moving Boundary Electrophoresis
: Charge particle moving towards boundaries and anode and cathode are not fixed. Ex: Protein separation especially from blood sample.
: In the following medium is semisolid in which different component are immobilized.
Horizontal Electrophoresis Vertical Electrophoresis
Supporting Media Used
• Cellulose acetate
• Agar Gel
• Acryl amide gel
A horizontal gel apparatus consist of a box which is divided into two components. Gel is palced on this plateform and buffer is added until tank is full. Electrode in each component supply in the electric field.
A typical vertical gel is cast in a cassette made up of two glass plates separated by spacers who run along the slide of the plate. The gel monomer solution is treated to initiate polymerization and poured in to the cassette. A comb is insert in to the top of the cassette to form the samples wells, and the gel is allowed sufficient time to polymerize.
Paper electrophoresis technique
is now little used, if the support medium is a filter paper the electrophoresis is carried out for 16 to 18 hrs. at a low voltage. Nowadays the preferred solid support media for horizontal electrophoresis is cellulose acetate membrane strip (Kohn 1957, 1976). They are expensive but the process takes less then one hours and and excellent separation without diffusion is achieved. This is widely used for separation and identification of Lipoproteins, Haemoglobins and Isoenzymes.
Other commonly employed supports are Agar or Agarose gel. It is most commonly used for Immunoelectrophoresis process by several years. This is the medium that adsorbs proteins very weekly and resulting as well defined bands which is suitable for Isoenzyme separation.
Starch gel electrophoresis (Smithies 1955) has all the advantage of other gels with the additional advantages of molecular sieving effect retarding protein of larger molecules molecular size. Its use now has been largely superseded by acrylamide gel .
Polyacrylamide gel electrophoresis (PAGE)
is most popular type of electrophoresis for molecular sieving effect. In agar electrophoresis, serum components are separated into five fractions while by PAGE technique serum will show more then 20 different bands. PAGE can be used either on Horizontal slab or vertical disc electrophoresis.
Acrylamide monomer (CH2=CH-CONH2) IS Polymerized by using an initiator and a catalyst to produce the polyacrylamide. The amount of crosslinking and pore size can be controlled by adjusting the concentration of the reagents.
Another common variant is the
SDS-PAGE (Sodium Dodecyl Sulphate PAGE) electrophoresis
. This technique is based on molecular weight of moving particles. Protein are boiled for 1-2 minutes with a denaturing agent, Sodium Dodecyl Sulphate and the negative charge of SDS will cover the protein molecules making them strongly negative.
ISOELECTRIC FOCUSING (ELECTRO-FOCUSING)
: A direct current passing through a series of ampholyte the pH of which increase gradually from anode to cathode. If pH gradient remains stable, protein and other ampholyte will be repelled from both electrodes and collect in region where the local pH is identical with the isoelectric point (pI). It has been mainly used as a research technique for separating proteins of closely similar structure such as isoenzyme and genetic variant of serum proteins and immunoglobulins.
: Electric separation is followed by an antigen-antibody reactions. Proteins are now separated to visualise them a specific antibody is placed in a through cut into the gel and incubated . Serum are fractioned into more then 40 bands therefore it is much more sensitive and specific then ordinary electrophoresis .
HIGH VOLTAGE ELECTROPHORESIS
: Since separation of molecules depends on the strength of current recent trend is to utilize high voltage (400 to 2000 volts instead of 250 volts). The advantage of this technique is result could be obtained within half an our. This technique now being widely used for separation of proteins, as well as nucleotides from biological fluids.
: In this type electrophoresis is carried out in a capillary tube. Very high voltages are used as the high surface to volume ration in the capillaries allow for good dissipation on the heat that is generated.
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