Citation Lee20 /L 1033

Electrophoresis
Introduction (Perias)
Have you ever been curious how do health care workers do tests for covid-19? Mainly, there are two
categories in the lab tests for detecting covid-19. These are the molecular and genetic tests that looks for
an infection.
Molecular tests are the ones we usually see practiced by the healthcare workers. On the other hand,
genetic tests look for the genes of the virus. Viruses' genetic code is stored in the RNA. A genetic test
makes the genetic code be copied in the DNA using reverse transcriptase. After that, the DNA is now
increased and boosted up to billions of copies using the polymerase chain reaction (PCR) process. To pick
out and separate the needed DNA sample , gel electrophoresis can be used. What happens is the
molecules are to be pushed by an electric field through a gel containing small pores. The molecules will go
through the pores in the gel which speed is determined by how small and big they are. [CITATION LEE20 \l
1033 ]
Figure 1. Agarose gel electrophoresis of the DNA amplification product created by targeting the sequence
coding for SARS-CoV-2 N protein.
Figure 2. Image of gel electrophoresis for RT-PCR products

Overview (Bagon, Pelagio)
Electrophoresis is a technique used to separate biomolecule such as DNA, RNA, or protein molecule based
on their size and electrical charge by subjecting it to a fluid medium. This technique is done by loading
sample molecule, for example DNA in wells of the agarose gel, the gel is then charge moving the sample
from one end to another, in this case a negative charged molecule moving toward the positive electrode.
DNA sample will be sifted by the sponge-like structure of the gel, separating them in sizes, the smaller one
travelling farther. This process allows patterns of As, Cs, Ts, and Gs to be detected by mixing fluorescent
intercalating dyes (e.g. EtBr) on the sample producing a bright orange bands of DNA under ultraviolet light.
[CITATION Chr21 \l 1033 ] [ CITATION Kha21 \l 1033 ] By proceeding with specific experimental conditions,
electrophoresis can be used to recognize and distinguish, purify, and quantify specific molecules.
[ CITATION Ann14 \l 1033 ]
Figure 3. Activity of Molecules in the Agarose Gel
Figure 4. Gel Electrophoresis Set up

Blotting (Bagon)
After gel electrophoresis, biomolecules are transferred to a membrane for further immunological or
biochemical analysis— blotting. These molecules are transferred to a membrane and immobilized to enable
radiolabeled or enzymatically labelled probes or antibodies to bind to the targeted biomolecule. [CITATION
MIC19 \l 1033 ] Different types of blotting for a specific biomolecule are: Southern Blotting for DNA,
Northern Blotting for RNA, and Western Blotting (immunoblotting) for protein. [ CITATION Ann14 \l 1033 ]
Table 1. Comparing Southern, Northern, and Western Blots.
Southern Blotting (Bagon)

Developed by Edward M. Southern in the 1970s, Southern Blotting is designed to detect DNA identity, size,
and quantity in the sample. It can also be used in identifying presence of mutated gene. This technique
rangers from confirming results of cloning experiments to forensic or clinical diagnostics. [CITATION Kri16 \l
1033 ]
Figure 5. Southern Blotting Technique

The process starts with collecting DNA sample and proceeds to enzyme digestion, a process where a
restriction enzyme scans the DNA sample for the targeted sequence and cut the strand. These sample will
undergo gel electrophoresis, after separation, double stranded DNA will proceed to denaturing to produce a
single stranded DNA that will be transferred to a membrane (e.g. nitrocellulose, nylon) done by capillary
action or a vacuum blot apparatus. Crosslink the DNA to the membrane by treating it under UV light to
immobilize. Pre-hybridization is required to block any non- specific binding in the membrane, done by
preparing a buffer added with the sample DNA. Remove the buffer and using single stranded probes, we
proceed to hybridization, annealing of the labeled probe to the DNA sequence targeted and then incubate
followed by a low to high stringency wash to refine DNA. Detection of probes are done autoradiograph (for
32P probe), Bioluminescent visualization or Colorimetric method (for biotin), Detected probes will determine
whether the sequence for the targeted DNA sequence exist in the sample. [ CITATION Kha10 \l 1033 ]
Advances made today made it possible to have easier automation of result with less time and resources.
Today, southern blotting has been largely replaced by real- time PCR, due to its efficiency to record
detected genes with lesser amount of DNA required, and less work. Though, southern blotting is still used
to detect transgenes in transgenic animals due to the lower false positive results in comparison to RT-PCR.
[ CITATION Kri16 \l 1033 ]
Northern Blotting (Perias)
Figure 6. Northern Blot Technique

Northern blots are used in knowing the specification, size, and abundance of an RNA sequence. Northern
blot protocols start with RNA isolation. RNAs are separated according to their sizes. Larger RNAs are
separated by gel electrophoresis that maintains RNA in its denatured state. Next is the electrophoresis. For
a formaldehyde agarose gel, put the tray with the gel inside the apparatus. Next is to load the samples and
don’t forget to include the molecular weight marker. Before blotting, run and trim the gel. For glyoxal
agarose gel, put the tray with the gel inside the apparatus. Load the samples into wells together with RNA
ladder. For denaturing polyacrylamide gel, put the gel in the electrophoresis unit and then prepare the
samples. After that, load and run with TBE running buffer. Third is to transfer. For a formaldehyde agarose
gel and glyoxal agarose gel, first wash the gel in SSC then the transfer unit must be assembled. It contains
the gel, filter paper, and nylon membrane. When transfer is finished, place it in a UV cross-linker. For
denaturing polyacrylamide gel, prepare the transfer unit containing gel, filter paper, and nylon membrane
and the must be flooded in TBE. When transfer is finished place it in a UV cross-linker. Fourth protocol is
the pre-hybridization and lastly is hybridization. Incubate the probe with the hybridization solution. To
remove, hybridization solution and unhybridized probe, wash the membrane very carefully. To remove
partially hybridized molecules, wash it with high stringency. Finally, follow the directions of the manufacturer
for chemiluminescent detection.[ CITATION MIC19 \l 1033 ]
Western Blotting (Pelagio)
Figure 7. Western Blotting Technique

Western blotting is a laboratory technique in molecular biology that is used to identify and analyze specific
proteins extracted from cells or tissue, through gel electrophoresis. Western blots used three elements
technique to detect the specific proteins. The separation by size, transfer to a solid support and marking
target protein using a proper primary and secondary antibody to visualize. The antibody is washed off
leaving only the bound to the protein of interest. The bound antibodies are then identified by building up the
film. As the antibodies bind to the protein of interest, one band will be visible. The thickness of the band
compares to the measure of protein present; consequently, doing a standard can demonstrate the measure
of protein present. [CITATION Tah12 \l 1033 ]
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) stimulates body reaction which can serve
as a marker for current or past exposure to this pathogen, and potentially for protection from re-infection.
This reaction to COVID-19 can be observed dependent on the production of antibodies, and along these
lines, serologic tests have opened for analytic purposes. Western blots is important to identify and analyze
antigens that could be reason for developing an antibody against COVID-19. To analyze this process would
be use skin testing to quantify the deferred type of touchiness reaction in patients recovering from COVID-
19. [ CITATION Pav20 \l 1033 ]
References
Austin M.D., C. P. (2021). Electrophoresis. Retrieved from National Human Genome Research Institute:
https://www.genome.gov/genetics-glossary/Electrophoresis#:~:text=Electrophoresis%20is%20a
%20laboratory%20technique,move%20faster%20than%20larger%20molecules.
Babtie, A. (2014). Electrophoresis and Blotting. London: Theoretical Systems Biology.
Don't Memorise. (2020, March 27). What is Gel Electrophoresis | Don't Memorise. Retrieved from YouTube:
https://www.youtube.com/watch?v=4OJAzQsZnbo
Dotzert PhD, M. (2019, September 29). Southern vs Northern vs Western Blotting Techniques. Retrieved
from Lab Manager: https://www.labmanager.com/insights/southern-vs-northern-vs-western-
blotting-techniques-854
Khalsa, G. ". (2010, April 12). Southern Blotting. Retrieved from Arizona State University School of Life
Sciences Ask A Biologist: https://askabiologist.asu.edu/southern-blotting#:~:text=Southern
%20blotting%20was%20named%20after,DNA%20within%20a%20complex%20mixture.
Khan Academy. (2021). Gel electrophoresis. Retrieved from Khan Academy:
https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis#:~:text=Gel%20electrophoresis%20is%20a
%20technique,move%20towards%20the%20positive%20electrode.
Lyon PhD, K. (2016, June 21). Evolution of Lab Techniques. Retrieved from addgene:
https://blog.addgene.org/evolution-of-lab-techniques
Pavia, C. S., & Wormser, G. P. (2020). COVID-19: Is there a role for Western blots and skin testing for
determining immunity and development of a vaccine? Diagnostic Microbiology and Infectious
Disease, 98 (4).
TESCHLER, L. (2020, March 19). How the health care system tests you for the Covid-19 virus. Retrieved
from testandmeasurementtips.com: https://www.testandmeasurementtips.com/how-the-health-
care-system-tests-you-for-the-covid-19-virus/
Yang, P.-C., & Mahmood, T. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North
American Journal of Medical Sciences, 429-434.

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Electrophoresis

Figure 2. Image of gel electrophoresis for RT-PCR products

Figure 3. Activity of Molecules in the Agarose Gel

Figure 4. Gel Electrophoresis Set up

Southern Blotting (Bagon)

Figure 5. Southern Blotting Technique

Northern Blotting (Perias)

Figure 6. Northern Blot Technique

Western Blotting (Pelagio)

Figure 7. Western Blotting Technique

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