While studying nucleic acids and proteins (DNA, RNA) in the lab, there is a huge chance that you will be testing your samples through gel electrophoresis. It is the most effective method for the separation of proteins, DNAs, and RNAs from a mixture. Hence, gel electrophoresis is a common instance in the streams of life sciences, food science applications, and mining. Vertical Gel Electrophoresis system and horizontal gel electrophoresis are the two types of gel electrophoresis systems.
What is vertical gel electrophoresis?
The technique of vertical gel electrophoresis system functions according to the theory of gel electrophoresis. However, it is more complex than the horizontal electrophoresis method. The vertical gel electrophoresis system uses a discontinuous buffer. A cathode is situated in the top chamber, and the anode is located at the bottom chamber. The electrodes in each compartment provide the required electric field. Thereafter, a thin layer of gel is applied between the two mounted glass plates.
The upper portion of the gel is submerged in the top chamber, and the bottom portion of the gel is positioned in the bottom chamber. When the current is applied, a small buffer portion migrates to the bottom chamber from the top chamber through the gel. The current implemented in this technique is of minute units.
Functioning of gel electrophoresis
Those who want to separate proteins and nucleic acids through this method should run their samples through a gel apparatus that comprises a cathode on one end and an anode on another. Besides these elements, there is also a platform that contains a porous gel matrix in the middle. This gel matrix is usually acrylamide gel or agarose. Now, a perfect buffer is added to form a charge gradient upon the implementation of electric current.
When the charge is applied, the gel will heat up, but the buffer helps to keep the gel cool and combat it from overheating. Both proteins and nucleic acids come with a uniformly net-negative charge. Hence, these molecules will move towards the positive electrode with the power supply. Since small molecules quickly move through the pores of the gel matrix, you can expect these molecules to migrate through the gel matrix quicker than the larger molecules. When the process is completed, you will come across distinct bands of proteins and nucleic acids separated grounded on their molecular weight.
Vertical vs. Horizontal electrophoresis: Understanding the differences
Broadly, there are two types of gel electrophoresis that you can implement for your application: vertical gel electrophoresis system and horizontal gel electrophoresis. Both systems operate on a similar theory of gel electrophoresis. However, there are also certain differences between the two. Let’s have a look:
Orientation and buffer system: One of the primary differences between the two systems is their orientation. When it comes to horizontal gel electrophoresis, the gel matrix is cast horizontally. Also, the matrix is submerged in a continuous running buffer. On the other hand, in the vertical gel electrophoresis system, the gel is vertically oriented with the buffer system being discontinuous.
Moreover, the vertical gel system uses two chambers—a top chamber and a bottom chamber. The cathode is present in the top chamber, and the anode is present in the bottom chamber. Electrodes are present in both chambers that produce the necessary electric field. Thereafter, a bit of gel is applied between two glass plates so that the gel’s bottom is submerged in a buffer in one chamber with the upper part being immersed in a buffer in another chamber.
When the electric current is applied, a small buffer amount moves down the chamber through the gel. You get complete control of voltage gradients as the buffer flows through the gel. This process highlights you can expect an effective separation and enhanced resolution when you implement the vertical electrophoresis system.
Gel: Vertical electrophoresis uses acrylamide gel while horizontal gel implements agarose gel. Generally, agarose gels have larger pores (up to 500 nm in diameter) while acrylamide gels come with smaller pores (up to 200 nm in diameter).
You can’t use acrylamide gel for horizontal gel electrophoresis. It is because in this method the gel is exposed to atmospheric oxygen. The presence of oxygen impacts the polymerization of acrylamide and interferes with gel formation. However, you have the chance to implement acrylamide gel in a vertical gel electrophoresis system as only the buffer flows through, and the specific compartments aren’t exposed to atmospheric oxygen.
When to implement either technique?
Generally, horizontal gel electrophoresis is used for separating mixtures comprising DNA and RNA molecules while a vertical gel electrophoresis system is implemented in separating proteins. When the task is about separating nucleic acids, horizontal systems are a popular choice due to their simplicity of use coupled with the ability to use the gel during the separation phase. However, while separating similar nucleic acids like dye termination sequencing, researchers generally opt for the increased resolution of a vertical system. Hence, the nature of the molecule being separated and the desired resolution of the gel determines which electrophoresis system is apt for application.
How can you generate reliable outcomes ?
Gel electrophoresis revolves around maximizing the operating conditions: buffer choice, voltage, dye, pH value of the buffer, and correct reference maker. All the above aspects can impact the resolution of the result of gel electrophoresis. You must know that the concentration of both agarose and polyacrylamide gel is crucial as they highlight the matrix’s pore size.
Besides this aspect, the voltage regulates the migration speed of the biological molecules provided that the molecules’ charge remains constant. Henceforth, it becomes easier for the researchers to manipulate the gel percentage and voltage to receive the optimal electrophoresis result.
Selecting the right buffer grounded on your experiment design is also an essential factor. The buffer choice doesn’t only limit the electrophoresis process, but its implementation is also a crucial aspect of picking the right buffer. While picking the reference maker irrespective of being protein ladder or nucleic acid, it’s important that you have a good idea of the product size you are expecting. If you use a ladder that falls outside the product range, the estimation of product size will become difficult.
Wrapping Up
Gel electrophoresis is a strategy implemented to separate DNA fragments (or macromolecules like RNA and proteins) grounded on their size and charge. This process involves running an electric current through a gel comprising the molecules of interest. Both horizontal and vertical gel electrophoresis systems are ideal choices for biological lab research. If you know the purpose that these strategies serve, you can easily identify the one to use to attain your research objectives.
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