Introduction and Principle

Horizontal electrophoresis tanks are generally used for the analysis and size separation of nucleic acids, i.e., DNA and RNA. However, it can also be used for the analysis of protein complexes. In a horizontal electrophoresis system, the gel matrix is cast horizontally and submerged in a running buffer within the electrophoresis tank. The tank is divided into two compartments separated by agarose gel. 

Horizontal electrophoresis tanks contain an anode at one end and a cathode at another. A charge gradient is created as the electric current flows through the ionic running buffer. The purpose of the running buffer is to cool the gel, which gets heated up as the charge is applied. The occasional recirculation of running buffer prevents the formation of a pH gradient. Unlike vertical electrophoresis tanks, the two chambers in horizontal systems are connected by running buffers and cannot be used with a discontinuous buffer system. 

Horizontal gel electrophoresis most commonly employs agarose gel. Agarose gels have larger pores, about 100-500nm in diameter. These gels help determine the size of the nucleic acids accurately based on their mobility through the gel. Acrylamide gel cannot be usually used for horizontal systems as the gel gets exposed to atmospheric oxygen in these systems, and oxygen prevents acrylamide polymerization, thereby interfering with gel formation. However, some modern electrophoresis protocols use polyacrylamide gel in horizontal systems.

Apparatus and Equipment

The horizontal electrophoresis system is made of acrylic and comprises two buffer chambers, each containing an electrode. The apparatus consists of a casting tray, horizontal electrophoresis tank (also known as a submarine tank), gel combs, electrodes, and a power supply. Conduct Science offers two different models of horizontal electrophoresis tanks. Each model has different sizes of casting trays and gel combs. 

Protocol

The underlying protocol is followed for a typical agarose gel electrophoresis for the resolution and recovery of large DNA fragments using horizontal electrophoresis tanks. 

Gel Preparation

1. Prepare a sufficient volume of electrophoresis buffer to prepare gel and fill the horizontal electrophoresis tank. The buffer used is usually TAE or TBE. Add 0.5µg/ml of ethidium bromide to the electrophoresis buffer to facilitate DNA visualization.
2. Mix an adequate amount of electrophoresis grade agarose with the buffer to construct the gel. Melt agarose and swirl to ensure uniform mixing. 
3. Seal the gel casting tray using tape, pour in the melted agarose, and insert the combs. Ensure no bubbles are trapped under the combs, and all bubbles on the gel surface are removed before the gel sets.
4. Remove the tapes once the gel hardens and withdraw the combs without tearing the wells. Most gel platforms allow 0.5-1mm of agarose to remain between the comb’s bottom and the base of the gel platform. This ensures that sample wells are completely sealed and prevents the tearing of wells while removing the combs. 
5. Place the gel casting tray containing set gel in the electrophoresis tank and add a sufficient volume of electrophoretic buffer to cover the gel up to 1mm deep. Avoid air pocket formation within the wells. 
6. Prepare the DNA samples in a volume that does not overflow the wells after adding 10x loading buffer. Use a micropipette to load the samples into the well. Avoid mixing samples. Include appropriate DNA molecular weight markers.
7. Attach the leads and set the voltage to the desired level (normally 1-10V/cm of gel) to run the gel. The dyes present in the loading buffer help monitor the migration of DNA fragments. 
8. Switch off the electricity supply when bromophenol blue from the loading buffer has moved a distance sufficient for the DNA separation. If EtBr is incorporated into the gel, you can directly visualize DNA under a UV source (Voytas, 2000).

Applications

Enhanced Detection of Protein-RNA Complexes

Dowdle et al. (2017) used horizontal polyacrylamide gel electrophoresis to analyze RNA-protein complexes at different points during the experiment. The researchers used Electrophoretic Mobility Shift Assay (EMSA) that employed polyacrylamide gel in horizontal/flatbed electrophoresis and fluorescently labeled RNA substrates. They analyzed 48 samples simultaneously using a horizontal electrophoresis tank equipped with a gel box (37cm × 24cm) and a gel casting tray (27cm × 21cm). Moreover, the tank could accommodate two 24-well combs. The researchers poured 2cm of gel into the casting tray, allowed it to sit for 30 minutes, removed the combs, and inserted this gel in the electrophoresis tank. The prepared reaction mix was loaded into the wells, and the horizontal electrophoresis tank was connected to the electric supply, the switch was turned on, and the gel was run at 120V and 4oC. Ultimately, the scientists observed the gel using a gel imager. They concluded that a few changes in the polyacrylamide gel electrophoresis method and adding fluorescently labeled RNA substrates could bring many advantages to molecular biologists.

Comet Assay 

Comet assay is a single cell gel electrophoresis assay that measures DNA strand breaks in eukaryotic cells. Sankar et al. (2010) studied the effect of curcumin on cypermethrin-induced genotoxicity in rats. The experimenters collected bone marrow cells from adult male Wistar rats (6 to 8 weeks old and 100–120 g in weight), separated peripheral blood lymphocytes from them, and performed a comet assay using a horizontal electrophoresis system. They placed the prepared slides in a horizontal electrophoresis tank containing a cold running buffer, i.e., 1mM Na2EDTA. Electrodes were connected, and the power supply was turned on. Electrophoresis was conducted for half an hour, following which slides were removed and examined for DNA fragments under a fluorescent microscope.

Succu et al. (2011) performed a similar assay to assess DNA integrity in an experiment while studying the effect of cryopreservation on ram spermatozoa. The agarose-coated slides containing 100µl sperm-agarose mixture were placed in the horizontal electrophoresis tank, and the tank was filled with TAE buffer. Electrophoresis was run at 10V and 6mA for about 20 minutes. The slides were neutralized, stained, and analyzed under an epifluorescence microscope for comet ‘tails.’

Strengths and Limitations

Horizontal electrophoresis systems present several advantages over vertical systems. For instance, in vertical electrophoresis tanks, a pump is required to push buffer from the lower chamber to the upper chamber, but horizontal gels do not require such pumps. Horizontal gels are prepared with larger wells that allow the researchers to analyze larger samples. Another advantage of horizontal systems is that they provide a higher resolution of protein-RNA complexes than vertical systems. Moreover, the gel can be imaged several times during the experiment. In addition, horizontal systems generate gels with a large number of wells compared to vertical format; hence, multiple samples can be run simultaneously (Dowdle et al., 2017). 

A potential disadvantage of horizontal electrophoresis systems is that the person can receive an electric shock if mishandled.

Precautions

• Ethidium bromide is a mutagen and a potential carcinogen. Always wear PPE like gloves, lab coats, etc., while working with it. 
• While preparing the gel, be careful to avoid air bubbles. Remove any bubbles appearing underneath the comb or on the gel surface to ensure the system’s efficiency.
• Always keep the apparatus covered and away from frequently used work surfaces to avoid electric shock.
• Do not leave the equipment unattended. 
• Do not expose yourself to ultraviolet light while performing gel imaging. 

Summary

• Horizontal electrophoresis tanks are generally used for the analysis and size separation of DNA, RNA, and proteins.
• The gel matrix is cast horizontally and submerged in a running buffer within the electrophoresis tank.
• Horizontal systems mostly utilize agarose gels; however, acrylamide gels can sometimes be used. 
• The horizontal electrophoresis system is made of acrylic and comprises two buffer chambers, each containing an electrode.
• The simplicity of use, cost-effectiveness, and ability to analyze a larger number of samples are advantages of horizontal electrophoresis systems.
• Horizontal electrophoresis tanks are frequently used to separate and analyze nucleic acids and comet assay.

References

Voytas, D. (2000). Agarose gel electrophoresis. Current protocols in molecular biology, 51(1), 2-5.

Dowdle, M. E., Imboden, S. B., Park, S., Ryder, S. P., & Sheets, M. D. (2017). Horizontal gel electrophoresis for enhanced detection of protein-RNA complexes. JoVE (Journal of Visualized Experiments), (125), e56031.

Sankar, P., Telang, A. G., & Manimaran, A. (2010). Curcumin protects against cypermethrin-induced genotoxicity in rats. Environmental Toxicology and Pharmacology, 30(3), 289-291.

Succu, S., Berlinguer, F., Pasciu, V., Satta, V., Leoni, G. G., & Naitana, S. (2011). Melatonin protects ram spermatozoa from cryopreservation injuries in a dose‐dependent manner. Journal of Pineal Research, 50(3), 310-318.