Gel electrophoresis is a powerful analytical separation technique particularly useful for biological analyses such as the analysis of proteins, nucleic acids (DNA and RNA), nanoparticles, etc. It operates on the same principle as that applied for any chromatographic technique. Therefore, gel electrophoresis is often considered a sub-type of chromatography with slight differences present. Thus, this article in our chromatographic series is solely dedicated to gel electrophoresis: its working principle, method, and applications.
What is gel electrophoresis
The word electrophoresis can be decomposed into electro and phoresis. Electro comes from electric current while phorus means carried by. Thus, electrophoresis is a technique specifically applied for the analysis of charge-bearing molecules, carried by an electric current. It is performed on a gel flatbed by applying an electric current thus the name gel electrophoresis is used. The charged particles migrate under the influence of the applied electric field and are separated on the basis of their shape, particle size, and charge.
Historical perspective of gel electrophoresis
Electrophoresis was observed for the first time back in 1807 by Ferdinand Frederic Reuss at Moscow State University. Gel electrophoresis properly however was introduced in 1937 by a Swedish chemist Arne Tiselius who later received a Nobel Prize in 1948 for his work on ‘Electrophoresis and discoveries concerning the complex nature of serum proteins’.
Working principle of gel electrophoresis
Gel electrophoresis works on an electrokinetic principle. A gel electrophoretic system is based on a stationary phase and a wet mobile phase in an electrolytic cell. Two electrodes of opposite charge called the cathode, and the anode are present, connected by an electrolyte. Negatively charged fragments from a complex sample mixture travel towards the positively charged anode and vice versa for positively charged fragments. Shorter molecules move faster and farther than the longer ones. In this way analytical separation takes place.
What is the stationary phase in gel electrophoresis
A porous gel matrix is used as a stationary phase in gel electrophoresis such as the agarose or polyacrylamide gel. The gel is a crosslinked (mesh-like) polymer whose composition and pore size can be varied as per sample requirement. The gel molecules are held together by hydrogen bonding which results in tiny pore formation in the gel.
Agarose gel has a large pore size, it is mainly used for the separation of nucleic acids and large proteins (larger than 200 kDa). The polyacrylamide gel is a more common stationary phase for the separation of small proteins via gel electrophoresis. Partially hydrolyzed starch can also be used as a gel medium for electrophoresis. In all cases, a horizontal plate is used for holding the gel.
What is the mobile phase in gel electrophoresis
Buffer solutions of varying pH are used as mobile phase solvents for sample preparation in gel electrophoresis. The tris-acetate-EDTA (ethylenediamine tetra acetic acid) and the tris-borate-EDTA buffer solutions are the most popular buffer choices in gel electrophoresis.
How to perform gel electrophoresis for a DNA separation
Step I: Gel packing
The agarose powder is first heated by adding the respective buffer. Upon cooling, a solid, squishy gel of agarose is formed. The electrophoretic plate is filled with this gel. Indentations called channels, lanes, or wells are created in the gel plate with the help of a fibrous comb.
Step II: Sample loading
The DNA sample prepared in a buffer solution is then loaded into these wells
Step III: Electric field application
An external electric field is applied to convert the electrophoretic system into an electrolytic cell. This turns the gel into a running medium.
Step IV: Analyte separation
The DNA molecules are negatively charged due to the negatively charged phosphate (PO34-) groups present in their structure. Thus, negatively charged DNA molecules migrate towards the positively charged anode. As the sample mixture consists of differently sized fragments of DNA so smaller molecules travel faster and farthest in the gel medium. The relatively larger molecules however stay behind. The separation of DNA fragments thus occurs in this way.
Step V: Identification
The fragmentation pattern obtained in step IV is compared to the standard fragmentation patterns available and the unknown sample is identified. The gel plate can also be stained with certain chemical visualizing agents to make the DNA fragments visible.
Ethidium bromide (C21H20BrN3) is often used as a visualizing agent. It intercalates into the DNA fragments and acts as a fluorescent tag so that the fragmentation pattern becomes visible under UV light as bands.
How to perform gel electrophoresis for a protein separation
Step I: Gel Packing
The electrophoretic plate is filled with the polyacrylamide gel. A pH gradient from 0-14 is set across the plate with the help of the buffer solution poured into the gel plate.
Step II: Sample preparation and loading
The protein sample is applied at the center of the gel-filled plate with the help of a micro-pipette.
Step III: Electric field application
An external electric field (approx. 100 volts) is applied, and the electrophoretic plate is converted into an electrolytic cell.
Step IV: Analyte separation
Proteins are macromolecules made up of a large number of amino acids. Charged amino acids in the protein structure lead to an overall charge present on the protein molecule. The positively charged protein molecules travel towards the negatively charged cathode while the negatively charged protein molecules travel towards the positively charged anode. When a protein reaches its isoelectric pH, it stops moving and is iso-focused. Thus, protein separation occurs.
The speed/ velocity (v) at which the charged protein molecules travel is the product of their mobility (m) and the applied electric field (E). v= m x E. The mobility (m) depends on particle shape, molecular size, charge, and temperature of the system.
Step V: Collection and quantification
The iso-focused protein molecules can be separately scratched out and extracted from the gel plate, collected, and quantified as a final step. Additionally, the gel as a whole can be transferred to a solid support and probed with specific antibody molecules for protein quantification. This method is known as Western blotting.
What is isoelectric focusing of proteins in gel electrophoresis
As we discussed already, proteins containing charged amino acids are called charged proteins. The isoelectric pH of a protein is the pH at which the polarity of different functional groups present on the protein molecule gets canceled. This gives the protein molecule a net zero charge. Thus, the protein stops moving on the gel plate. This phenomenon is called isoelectric focusing or iso-focusing.
How to calculate the isoelectric pH of a protein molecule
A protein molecule based on three different amino acids i.e., glycine, lysine, and aspartic acid is given. The amino acids consist of charged carboxyl and amino functional groups. All these groups have a specific pKa value.
To calculate the isoelectric pH of the protein :
Step I: Arrange all the pKa values given in an ascending order.
Step II: Choose four different pH values such that one pH value is lower than the lowest pKa. One pH is higher than the highest pKa, and three pH values are such that they lie in between pKa1, pKa2 pKa3, and pKa4 respectively. Refer to the supporting figure given below so that you may have a better idea.
Step III: Calculate the net charge present on the protein molecule at each pH selected.
Step IV: Note the pH at which the net charge calculated is equal to zero. Take the average of the two pKa values in between which this pH lies. This is the isoelectric pH for the protein i.e., the pH at which it will be iso-focused on the electrophoretic gel plate.
In case if a sample mixture is initially composed of uncharged protein molecules then it can be treated with a detergent such as sodium dodecyl sulfate. This chemical treatment unfolds the tertiary structure of the protein molecules into a linear shape, coating them with negative charges to allow their separation via gel electrophoresis.
Gel electrophoresis for protein separation is usually performed in one dimension. On the other hand, DNA fragmentation through gel electrophoresis is often performed as a two-dimensional protocol. 2-D electrophoresis allows faster separation of molecules in large bulk.
How is gel electrophoresis different from other types of chromatography
- A horizontal plate is used for holding the stationary phase in gel electrophoresis while the stationary phase in other types of chromatographies is held on a vertical plane such as in paper chromatography or packed into a column in column chromatography.
- In all other chromatographies, the mobile phase travels under the influence of gravity or pressure is applied such as in HPLC. In-gel electrophoresis, the analyte molecules migrate under the influence of an applied electric field.
Applications of gel electrophoresis
- Gel electrophoresis is very valuable to the forensic industry. Recognizing DNA fragmentation patterns can help resolve crimes.
- Nucleic acid electrophoresis is widely applied for genome sequencing and DNA fingerprinting. It can also be used for parental recognition.
- Gel electrophoresis is used by microbiologists to study and invent new drugs, and gene therapies.
- Gel electrophoresis can be used in medical diagnosis and for the detection of food contaminants.
- Protein-specific electrophoresis can be used in immunology.
Limitations of gel electrophoresis
- Passage of electricity produces heat which may melt the gel, changing its composition.
- Slight temperature fluctuations lead to variations in the rate of migration of charged particles through the gel medium. This may cause inaccuracies in the overall results obtained.
- The charge present on particles and their migration depends upon the pH of the buffer solution. Running a gel electrophoretic system for too long may exhaust the solution’s buffering capacity thus leading to inaccuracies in the results.
Considering the versatile applications of gel electrophoresis in multiple scientific fields, we conclude that it is a simple analytical tool that allows rapid separations with high sensitivity.
Here is a video tutorial for you on gel electrophoresis to revise all the concepts you learned in this article.
Also, check out this scrollable interactive to learn about DNA fragment separation through gel electrophoresis in a more fun way.
For more in-depth information on the different types of gels and running buffer solutions used in protein electrophoresis, you may like: Overview of protein electrophoresis.
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2. Magdeldin, S. (2012). Gel Electrophoresis: Principle and Basics.
3. Michael.J.Dunn (2014). Gel electrophoresis of proteins, 1-141.