Basic of Electrophorosis (An Emerging Tool For Bio – Medical Researchers)
The phenomenon of the migration of charged particle in the influence of electrical field is termed as Electrophoresis. It is one the powerful means of separating proteins and other bio-molecules such as DNA, RNA etc.
PRNCIPLE & THEORY:
When a potential difference is applied across the electrodes , a potential gradient will be generated which is equal to the ratio of potential difference and the difference the two electrodes.
Potential Gradient (E) = V / f
Where:
V = Volatage applied.
f = Distance between the two electrode.
For example:
If potential difference of 100 V is applied and the distance between the two electrode is 20 cm then:
Potential Gradient = 100 / 20 = 5 V/cm
When potential gradient is applied , the force acting upon the charged molecule having charge q will be:
F = E.q
So, the charge molecule will move with this force, but there is some frictional resistance that slow down the movement of this charge molecule i.e. size and shape of this charge molecule , pore size of the medium in which electrophoresis taking place and the viscosity of the buffer. . . . velocity of charged particle in an electrical field = F/f
= E.q /f
Now, the current in the solution between the electrodes is conducted mainly by the buffer ions.
Than according to Ohm’s law:
R = V / I
or applied voltage is directly proportional to flow of current.
Above statement demonstrate that it is possible to accelerate an electrophoratic separation by increasing the applied voltage , which will result in a corresponding increase in the current flow.
The distance migrated will be proportional to both current and time.
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However, increasing the voltage will cause generation of heat which have the following effects:
- An increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples.
- Formation of convection currents, which leads to missing of separated samples.
- Terminal stability of samples that are rather sensitive to heat. They may include denaturation of protein.
- A decrease of buffer viscosity, and hence a reduction in the resistance of the medium.
Factors that influence electrophoresis:
- Longer electrophoresis run will increase the separation between fragments . Adequate separation is important for analysis of DNA fragments, especially those that are close in size.
- The higher the voltage , the faster the DNA will be travel through the gel or cause smearing or distortion of DNA bands.
- The gel concentration and volume (thickness ) affects electrophoretic separation.
For exp. – DNA samples will migrate faster in 0.8 % gel compared to a 1% gel likewise. Samples will migrate faster in a 20 ml (6 mm thick ) gel verses a 30 ml (8 mm thick ) gel with the same 7 × 7 centimeter dimension.
- 4 )Electrophoresis through agarose or polyacrylamide gels is the standard method used to separate, identify and purify DNA fragments.
T5) his technique is simple, rapid to perform and capable of resolving fragments of DNA that can not be separated adequately by other procedures, as density gradient centrifugation.
