Agarose gel electrophoresis
Agarose gel electrophoresis is the common way of separating and analyzing DNA biomolecules such as DNA and RNA. It is an analytical method for determining the size of DNA molecules in the range of 500 to 30,000 base pairs. DNA bands visualization can be done by the addition of Ethidium bromide in the agarose gel. As DNA is negatively charged molecule, it moves from (cathode) negative electrode towards positive electrode (anode). The medium to be used for separation is a gel made from agarose
The migration of DNA depends upon many factors in agarose gel like:
1. Concentration of Agarose: Different concentrations of agarose gel separate different sizes of DNA fragments. With higher concentrations of agarose (1.5%), separation of small DNA‘s are done efficiently and with low concentrations of agarose (0.7%), separation of larger DNA‘s are done more efficiently. This is due to the fact that with the increasing concentration the pore size decreases which retard movement of the large molecular weight DNA fragments while for the separation of smaller molecules higher concentration gel is used.
2. Voltage: It also plays a very important role in gel electrophoresis. With increase in voltage applied to gel, larger fragments migrate proportionally faster than small fragments.
3. Electrophoresis Buffer: Buffers maintain pH (8.0) and provide ions to support conductivity. Several different buffers have been recommended for electrophoresis of DNA. The most commonly used are Tris-acetate EDTA (TAE) and Tris-borate-EDTA (TBE). These two buffers differ in ionic strength so DNA fragments migrate at different rates in these two buffers. TBE is a better option when analytical gel is run as the borate ions are more stable during a long duration run.
4. Ethidium Bromide: It is a fluorescent dye that intercalates between bases of nucleic acids and allows easy detection of DNA fragments in gels. With the binding of Ethidium bromide to DNA, mass and rigidity of DNA is altered which effects mobility as well and it can be added to agarose gel directly or it can be added to samples of DNA before loading to enable visualization of the fragments within the gel. Ethidium bromide fluoresces under UV light at 260-280nm which is the wavelength at which DNA absorbs light.
Two Dimensional (2-D) Gel Electrophoresis
This method is mainly employed for the separation and identification of proteins by displacement in two dimensions. The first dimension is isoelectric focusing and second dimension is SDS-PAGE. It is the only method that is capable of separating thousand of proteins simultaneously.
Isoelectric focusing: It is an electrophoretic method that separate proteins according to their isoelectric points and is ideal for separation of amphoteric substances. Isoelectric point is the point at which net charge on molecule is zero. The gels that are used in this technique are agarose gel and polyacrylamide gel. The separation is achieved by applying a potential difference across a gel that contains a pH gradient from highly acidic to highly basic pH.
In this technique, ampholytes are used in buffers to smooth the pH gradients. These are the complex mixtures of synthetic polyamino-polycarboxylic acids. Resolution in this technique depends on many factors like- pH gradient, thickness of the gel, time of electrophoresis, applied voltage etc.
On this gel, the protein molecules are separated on the basis of their charges and even one charge variation gives separation of the molecules. This strip of gel with bands separated on the basis of charge is then laid on SDS-PAGE for separation on the basis of mass.
Polyacrylamide Gel Electrophoresis (PAGE):
It is used to separate proteins according to their molecular weight. In short, single-stranded DNA molecules like oligonucleotides are well separated on polyacrylamide gel whereas larger DNA molecules (more than 100 bp in length and double stranded DNA molecules) are best resolved on agarose gels. SDS-PAGE uses an anionic detergent (SDS) to denature proteins and the protein molecules become linearized. Due to this the charge to mass ratio of all the denatured proteins in the mixture becomes constant. On an average, one SDS molecule binds to two amino acids. These protein molecules move in the gel towards the anode on the basis of their molecular weights. The gel system is formed of polyacrylamide. The polyacrylamide chains are crosslinked by N, N-methylene bisacrylamide monomers. Polymerization is initiated by ammonium persulfate which act as radical source and catalysed by TEMED which acts as a free radical donor and acceptor. This gel electrophoresis system is a discontinuous gel system comprising of two gels; Stacking gel (pH 6.8, %T = 3 to 5 %) and Separating gel (pH 8.8, %T = 5 to 20 %). At pH = 6.8, most of the glycine in the population exist as zwitter ions with no negative charge so bulk of the current is carried by the denatured and negatively charged SDS-coated protein molecules.
At this stage, the glycine ions lag behind the proteins. The order is as follows chloride ions, denatured proteins, glycine ions. On entering the resolving gel (pH=8.8), the glycine zwitter ions gets deprotonated to the anionic form. The carrying of the current is now shared by the ions such that protein molecules have a greater freedom to separate on the basis of molecular weights. Due to the small size of the glycine anions, they also tend to overtake the protein band, thus provide a sandwiching effect and greater resolution in the gel.