Gel Electrophoresis is a technique used to separate DNA, RNA or protein molecules based on their size, electrical charge and conformation using electric field at a definite pH.
It is based on the principle that charged molecules when placed in an electric field, tend to migrate towards the electrode of the opposite charge.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species, either cations (positively charged) or anions (negatively charged).
Gel Electrophoresis makes use of porous gels that act as a molecular sieve separating bigger molecules from the smaller ones.
The rate at which each molecule travels through the gel is called its electrophoretic mobility and its determined mainly by its net charge, size & shape.
- Strongly charged molecules move faster than weakly charged ones.
- Smaller molecules move faster than larger ones.
- Molecules with highly coiled structures move faster than uncoiled ones.
Nature of charge:
- Under the influence of an electric field these charged particles will migrate either to cathode or anode depending on the nature of their net charge.
- Nucleic acids which are negatively charged, due to the presence of the sugar-phosphate backbone, migrate toward the anode which is positively charged.
- Amino acids that make up proteins may be positive, negative, neutral, or polar in nature depending on the functional groups present in the amino acid. The charges of amino acids together give a protein its overall charge.
- Thus, when researchers want to separate proteins using gel electrophoresis, they must first mix the proteins with a detergent called sodium dodecyl sulfate (SDS). This treatment makes the proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward the positive end of the gel and be separated.
Voltage:
When a potential difference (voltage) is applied across the electrodes, it generates a potential gradient (E), which is the applied voltage (V) divided by the distance “d” between the two electrodes i.e.
| E = V/d |
When this potential gradient ‘E’ is applied, the force as the molecule bearing a charge of ‘Q’ is
| F = E x Q |
Unit of Force (F) is Newtons & Unit of Charge (Q) is coulombs
It is this force that drives the molecule towards the electrodes.
Frictional force:
There is also a frictional force that retards the movement of this charged molecule.
- This frictional force depends on :
- Hydrodynamic size of the molecule
- Shape of the molecule
- Pore size of the medium in which the electrophoresis is taking place
- Viscosity of the buffer.
The velocity ‘v’ of the charged molecule is an electric field is therefore given by the equation :
| V = (E x Q)/f =(V x Q)/(d x f) |
where ‘f’= frictional coefficient
Electrophoretic mobility:
Electrophoretic mobility ( u ) of an ion is represented as the ratio of the velocity of the ion and the field strength. i.e.
| u =V/E |
When a p.d. is applied, the molecule with different overall charges will begin to separate owing to their different electrophoretic mobility.
Even the molecule with similar charges will begin to separate if they have different molecular sizes, since they will experience different frictional forces.
Current:
According to Ohm’s law:
| V/I=R |
Therefore, it appears that it is possible to accelerate an electrophoretic separation by increasing the applied voltage, which ultimately results in corresponding increase in the current flowing.
The distance migrated by the ions will be proportional to both current and time.
Heat:
One of the major problems for most forms of electrophoresis, that is the generation of heat.
During electrophoresis, the power ( W ) generated in one supporting medium is given by
| W= I2R |
Most of the power generated is dissipated as heat.
The following effects are seen on heating of the electrophoretic medium has :
- An increased rate of diffusion of sample and buffer ions which leads to the broadening of the separated samples.
- Formation of convention currents, which leads to mixing of separated samples.
- Thermal instability of samples that are sensitive to heat.
- A decrease of buffer viscosity and hence reduction in the resistance of the medium.
If a constant voltage is applied, the current increases during electrophoresis owing to the decrease in resistance and this rise in current increases the heat output still further.
For these reasons, often a stabilized power supply is used, which provides constant power and thus eliminates fluctuations in heating.
Constant heat generation is however a problem. For which the electrophoresis is run at very low power (low current) to overcome any heating problems, but this can lead to poor separation as a result of the increased amount of diffusion due to long separation time.
Compromise condition have to be found out with reasonable power settings, to give acceptable separation time and an appropriate cooling system, to remove liberated heat. While such system works fairly well, the effect of heating are not always totally eliminated.
Electroendosmosis:
Electroendosmosis occurs due to the presence of charged groups on the surface of the support medium.
For instance, paper has some carboxyl group present, agarose contains sulfate groups depending on the purity grade and the surface of glass walls used in capillary electrophoresis contains silanol (Si-OH) groups.
These groups, at appropriate pH, will ionize, generating charged sites.
It is these charges that generate electroendosmosis.
In case of capillary electrophoresis, the ionized sianol groups creates an electrical double layer, or a region of charge separation, at the capillary wall/electrolytic interface.
When voltage is applied cations in the electrolyte near the capillary walls migrate towards the cathode, pulling electrolyte solution with them.
This creates a net electro osmotic flow towards cathode.
Jatayo 