Electrophoresis refers to the phenomenon of separation of charged molecules in the presence of the applied electric field. This process is also known as gel electrophoresis because the separation of charged species is done by using gel materials. This is a standard method to separate, identify and purify biomolecules such as nucleic acid, proteins, etc.
How do biomolecules separate? We know that most biomolecules such as amino acids, peptides, proteins, nucleotides, and nucleic acid possess certain functionalities, and hence, at a given pH these bear either positive or negative charge depending on the functional group. As a result, electrically charged species known as cations or anions can exist in the solution. When these charged molecules are introduced into an electric field, they start to move towards the electrode of opposite charge as a result of electrostatic attraction. Before explaining the separation process, let’s discuss the basic principle of gel electrophoresis.
Principle of Gel Electrophoresis
If we introduce a mixture of electrically charged biomolecules in an electric field(E), these will move freely towards the electrode of opposite charge. Positively charged molecules will move toward the negatively charged electrode and negatively charged molecules will move toward the positively charged electrode due to the electrical force of attraction. However, the rate of movement of different molecules toward electrodes will vary depending on the physical characteristics of the molecules. In addition, the experimental setup also affects the movements of biomolecules.
Indeed, the velocity(V) of movement of a charged biomolecule in an electric field(E) depends on various factors which can be explained by following mathematical expression.
where q is the net charge on the molecule which is determined by the number of positive and negative charges in the molecule, and f is the frictional coefficient. The frictional coefficient is a parameter that is used to describe the frictional resistance to the mobility of molecules and depends on a number of factors which are listed below.
- mass of molecule
- degree of compactness or molecular shape of the molecule
- the viscosity of the buffer solution
- the porosity of the matrix (gel) in which the experiment is performed
From the above equation, we can generalize that molecules will move faster with an increase in their net charges, an increase in the strength of the applied electric field, and as the frictional coefficient(f) decreases.
If two different biomolecules have similar net charges on them, how does the electrophoresis technique separate them? Actually, molecules with similar net charges separate due to differences in frictional coefficient while molecules of similar mass/shape may differ widely from each other in net charge. Consequently, it is possible to achieve very high-resolution separation by electrophoresis.
Every electrophoresis procedure is carried out in a suitable buffer. Any change in pH will affect the net charge and change the mobility of molecules.
Problems of electrophoresis
During experiments, an electric field is generated by applying the voltage V to a pair of electrodes separated by a distance d. One of the main issues with electrophoresis is heat generation that heats in the electrophoretic medium and it causes the following problems.
- Heat will increase the rate of diffusion of the sample and buffer ions which leads to the broadening of separating samples.
- The generated heat may denature heat-sensitive biomolecules.
- Convection currents may be generated which may mix separated samples
Diagram of gel electrophoresis
The schematic diagram of gel electrophoresis is shown below:
Common Support media in gel electrophoresis
Initially, electrophoresis was performed in free solution by Arne Tiselius who won the noble prize in Chemistry in 1948, which had many problems of diffusion and convection currents associated with this approach. But later, all these problems were resolved by performing experiments on porous supporting media.
The introduction of the use of gels as a supporting medium has improved the efficiency of separation. The commonly used support media in gel electrophoresis nowadays involve either agarose gels or polyacrylamide gels.
Agarose is a linear polysaccharide having an average relative molecular mass of about 12000. It is a polymer of agarobiose that comprises alternating units of galactose and 3,6-anhydrogalactose. It is one of the components of agar that is a mixture of polysaccharides isolated from some seaweeds.
The intramolecular and intermolecular hydrogen bonding inside and between the lengthy agarose chain is responsible for gelling properties of this matrix. Because of its cross-linked structure, the gel has effective anticonventional capabilities.
The repeating unit of agarose, agarobiose is shown below.
The pore size of the agarose gel can be controlled by the initial concentration of agarose. It means if we use a low concentration of agarose there will be large pore sizes formed and smaller pore sizes are formed from the higher concentration.
- Agarose is commercially available in the market in different purity grades based on the sulfate concentration. The sulfate concentration present in the gel plays an important role in the purity, the lower the sulfate content higher is the purity. The presence of sulfate causes electro-endosmosis during electrophoresis and ionic interaction between the gel and sample, both are considered as unwanted effects.
- Agarose gels can be used for the electrophoresis of both proteins and nucleic acid but are mostly used for nucleic acid. However, for the separation of proteins pore size of gels must be large. Usually, 1% agarose gel is used for this purpose.
Polyacrylamide gels are formed by the polymerization of acrylamide monomers in the presence of smaller amounts of a cross-linking agent N, N’-methylene-bisacrylamide.
Acrylamide gels can be made with a content of between 3% and 30% acrylamide. Therefore, low concentrations of gels have large pore sizes and are used for the separation of large size proteins molecule so that proteins can move without any frictional effects.
Low-percentage acrylamide gels are also used to separate DNA. Gels of between 10% and 20%
acrylamide is used in techniques such as SDS–gel electrophoresis, where the smaller pore size now introduces a sieving effect that contributes to the separation of proteins according to their size.
Types of gel electrophoresis
On the basis of support media used in the electrophoretic experiments, there are basically two types of gel electrophoresis.
- Agarose gel electrophoresis
- SDS-Polyacrylamide gel electrophoresis
Besides these, there are the following types of other electrophoretic techniques being used.
- Capillary gel electrophoresis
- Two-dimensional polyacrylamide gel electrophoresis(2d-gel electrophoresis)
- Cellulose acetate electrophoresis
- Pulsed-field gel electrophoresis
- Microchip electrophoresis
Gel electrophoresis protocol
Generally, the genomic DNA and proteins are examined through electrophoresis, and the following protocol is used to perform this technique.
- Initially, the DNA samples isolated from phenol: chloroform method is dried completely to remove excess ethanol and moisture.
- After the complete drying of ethanol, about 20-30 microlitres of TE buffer( Tris-EDTA) is added to each sample.
- Then, the working solution of DNA is prepared by adding 3 microlitres of DNA sample along with 1 microlite of 6X loading dye ( bromophenol blue).
- About 3-4 microlitres of the above mixture are pipette out and loaded in the gel well carefully.
- Likewise same as the sample, the ladder(1Kb) is prepared by adding TE buffer, dye, and ladder, and about 1.5 microlitre of the ladder is pipette out and loaded into the well.
- After complete loading, the positive and negative terminal is connected to the poles of the gel tray and connected to the power supply.
- The gel electrophoresis is performed at 75 volts, 40 amperes current, and run for about 1 hour after the complete run of gel, the DNA band and intensity are observed in Gel Doc ( UVITEC, Cambridge).
Gel electrophoresis buffer
For the buffer, we generally use 1X TAE ( Tris-acetate EDTA ) buffer to fill the gel tray for the separation of nucleic acids. TAE buffer serves the dual purpose of facilitating nucleic acid mobility through the agarose matrix and ensuring electric current passes through the gel.
Gel electrophoresis comb
A gel electrophoresis comb is a small comb-like tool made from plastic and it is used to make sample loading wells in the gel so that DNA/RNA or proteins can be placed. Therefore, it is an important tool in electrophoresis.
Gel electrophoresis ladder
A gel electrophoresis ladder is a mixture of known fragments of DNA/RNA (or protein) which is used as a reference to determine the size of the fragments of DNA/RNA under experiments or tests.
During experiments, the ladder is also run parallelly with the test sample on the gel. The size of fragments in the test sample is then determined by comparing the distance covered to those of fragments in the ladder.
Types of gel electrophoresis ladder
The gel electrophoresis ladders are commercially available in three different types viz. DNA ladder, RNA ladder, and Protein ladder. Basically, these have the same utility in electrophoretic experiments to estimate the size of DNA/RNA or molecular weight of proteins.
- DNA ladder
- RNA ladder
- Protein ladder
Errors in gel electrophoresis
As we know, error affects the accuracy of our result. Thus, we must know the source of errors so that we can eliminate these factors. Some major sources of errors in gel electrophoresis are listed below:
- Error due to improper sample preparation
- Overloading or underloading of sample
- Variation in Buffer composition, pH, temperature, etc.
- Error due to overstaining/understanding
- Error during the preparation of loading wells
Advantages of gel electrophoresis
In molecular biology and its related field, gel electrophoresis has been established as an important technique for the separation and analysis of DNA, RNA, and proteins according to their size and charges.
The major advantages of this technique are listed below:
- The first and most important advantage is its separation capability. This method can be used to separate biomolecules using a gel matrix which acts as a molecular sieve thus smaller biomolecules easily move while larger one’s movement is hindered due to their incapability to enter through the pore of the matrix.
- The molecular mass of biomolecular mass can also be determined by using this technique.
- This technique allows us to quantify biomolecules on the basis of the intensity of the band.
what is the purpose of the ladder in gel electrophoresis?
The ladder is used as a reference in gel electrophoresis to determine the size of the fragments of DNA/RNA under experiments or tests.
what is the comb used for in gel electrophoresis?
The comb is used for making sample loading wells in gel electrophoresis.
what is the purpose of ethidium bromide in gel electrophoresis?
Ethidium bromide is a cyclic planer molecule that binds between the stacked base pairs of DNA and thus under UV light, it fluoresces orange-red which helps in the visualization of DNA/RNA bands.
what is the purpose of loading dye in gel electrophoresis?
The purpose of loading dye in gel electrophoresis is to visualize the separated DNA bands.
what is the purpose of the buffer in gel electrophoresis?
The purpose of the buffer in gel electrophoresis is that it helps in the maintaining of pH of the solution as well as it helps to conduct electricity during the experiment.