Jason Bao

Sappington
AP Biology – Period 3
15 January 2021

Virtual Western Blotting Lab

Website: https://aelp.smartsparrow.com/v/open/zwzespn2
1. What is a Western Blot? What is it used for?
Western blotting (WB) is a common and important laboratory technique that is used to identify or characterize proteins
from a complex mixture of proteins according to their size and amount. Essentially, it is used to determine protein
expression.
2. What are the 3 stages of a Western Blot?
The three stages of a Western Blot are gel electrophoresis to separate proteins according to their size, blotting to transfer
proteins from the gel onto a support membrane, and antibody detection and visualization to locate the proteins of interest.
3. What impact does the pore size have in a gel?
The pore size in the gel determines how rapidly proteins of a certain size can migrate through the gel. Pore size is
determined by the percentage of acrylamide in the mixture.
4. What is a plate sandwich? How is it prepared?
A plate sandwich is a glass plate used to cast the protein gels that will be used in Western blotting. It is prepared by
putting two plates together, inserting this into a gated system, securing the plates into the gate system by pushing back the
gates, placing the glass-plate system into a larger gate system on top of the rubber base, and finally securing the gated
system.
Follow the instructions on the slide show as it instructs you to click on the top blue button to complete the virtual lab.
Follow instructions. When you have finished, hit Next at the bottom.
5. Explain what the loading buffer is and what the purpose of each thing is.
A loading buffer is a solution added to a gel electrophoresis sample to give it color and density. It contains glycerol to
increase the density of a sample and help it sink to the bottom of the well, bromophenol blue or other dye to keep track of
how far proteins have migrated in the gel, a reducing agent to break disulfide bonds and linearize a sample (run down the
gel according to size and not shape), and SDS to coat the protein in a negative charge so that the protein will migrate to
the positively charged end according to size and not charge. Thus, a loading buffer enables equal loading so that protein
levels and expression differences to be accurately quantified in western blotting.
Follow the instructions in the slide show, watch the videos, and then load your own virtual gel.
6. How difficult was it to load the gel? What could impair you in real life when you do this?
It looked relatively difficult to load the gel from viewing the video. In real life, the position of entry that the tip enters the
gel, ensuring that the tip is in the center of the well, and slowly loading at a steady pace so that the material is loaded
evenly are all challenges that can impair you when you load the gel in real life.
7. What is the process by which you can visibly see the results from the Western Blot?
The process to visibly see the results from the Western Blot involves blocking the membrane of proteins with 5% skim
milk powder proteins to prevent the antibodies used in subsequent steps from binding non-specifically to the membrane,
then incubating this blocked membrane with a primary antibody that is specific for the target protein, then washing and
incubating the membrane a secondary antibody that binds specifically to the primary antibody, then incubating the
membrane with chemiluminescent reagents that activate the reporter enzyme bound to the secondary antibody to detect
the specific binding of the primary antibody to the target protein, and then finally activate the reporter enzyme by placing
a X-ray film on top of the membrane in a dark room, where a banding pattern develops and thus the results from the
Western Blot can be seen.

The generalized steps to a Western Blot are seen here:

The locations and width of the band that occurs after step 4 allows us to see how much protein is present or has been
produced from a gene. This allows us to monitor translation or gene expression levels.
On the next page, you can see an example of a Western Blot result. The scientists in this lab most likely used computer
software to analyze the size of the bands to determine the actual protein amount they amount to (though there are methods
to doing this by hand).
As you can see, there is a pretty big result in the amount of protein result in the variable group for P11 but not p33 from
the control.
Now, watch the following video. Don’t worry, it’s short. https://www.youtube.com/watch?v=xNcXVpX6iyY
8. Compare Northern, Southern and Western blots with at least 3 points of comparison between them.
Overall, Southern blotting is used to detect the presence of a particular DNA fragment in a sample, Northern blotting
is used to detect specific RNAs (mRNA), and Western blotting is used to detect specific proteins, so the three methods
differ by what macromolecule they are detecting. Additionally, while both Southern blotting and Northern blotting rely on
the principle of hybridization (using a radio labeled DNA or RNA probe to find out the target DNA/RNA to bind
specifically in a target and form a hydrogen bond that can be easily recognized), Western blotting relies on the principle of
antigen antibody interaction in which a primary and labeled antibody are added to a highly specific protein considered as
an antigen so that the antibody can bind to the specific antigen, causing signals to be released if the protein of interest is
present. Moreover, each blotting technique was created by different scientists – Southern blotting by Edward Southern in
1975, Northern blotting by Alwine and his colleagues in 1979, and Western blotting by Towbin in 1979. Finally, while
both Southern and Northern blots use nylon membranes to transfer DNA or RNA from the polyacrylamide gel, Western
blots use a nitrocellulose or PVDF membrane to transfer proteins. However, all three blotting methods rely on
electrophoresis for fragment separation (DNA, RNA, or proteins), a similarity between the three distinct methods.
On the next page is another example of results. They go from left to right and on the right you can finally compare the two
tables to see protein expression differences between a neonatal infant and an adult.

9. Think! Propose a GENETIC reason for why the protein expression of a neonatal infant can differ from that of an
adult. Use a specific example from the image above to support your claim.
 A genetic reason for why the protein expression of a neonatal infant can differ from that of an adult is the presence
and activation of certain epigenetic markers. Whether through chemical (methylation of DNA) or protein (acetylation of
histones) epigenetic markers, certain genes can be turned on or turned off, causing the neonatal infant to differ in protein
expression than an adult due to differences in activated genes. In other words, before the infant is born, though the genetic
code remains unaltered, environmental factors like diet, stress, and prenatal nutrition can make an imprint on genes passed
from the parent to the infant through epigenetic marks, causing differences in protein expression. For example, in the
image, in respect to spinal cord protein expression, while an adult had 605 a.u., a neonatal infant had 583 a.u., a difference
which could have resulted from the repression of certain genes by the deacetylation of histones that prevents spinal cord
expression proteins from being expressed, thus creating the difference in protein expression in respect to amounts of
protein (a.u.) seen in the image. The alternative is also true in that in regards to kidney protein expression, neonatal infants
could contain genes that were not methylated as they were in adults, causing these genes and resulting proteins to be
expressed, causing neonatal infants to have greater protein expression in their kidneys.
10. Finally, write a 4 sentence minimum summary of what you learned regarding blotting.
Overall, regarding blotting, I learned that blotting is a crucial laboratory technique to identify proteins and nucleic
acids from a complex mixture by their respective differences in size and amount. Specifically across the three main
methods of Southern, Northern, and Western blotting, the three general stages are separating the DNA, RNA, or proteins
by size, blotting to transfer the DNA, RNA, or proteins from the gel onto a support membrane, and then using either
hybridization or antibody detection and visualization to locate the proteins or nucleic acids of interest. Additionally, I
learned that computer software is often used to analyze the size of bands of the blots to determine actual protein amounts
for comparison, but there are methods for doing this by hand albeit at a much slower pace. Moreover, I learned that a
loading buffer used in the blotting process is added to a gel electrophoresis sample to give it color density. Because the
buffer contains glycerol, a type of dye, a reducing agent, and SDS, it ensures that the protein or nucleic acid will sink to
the bottom of the well and that the migration of both are clearly visible, which enables the differences to be accurately
quantified. Finally, I learned that Western blotting can be used to explicitly and quantitatively compare differences in
protein amounts that result from changing gene expression levels in variable groups of an experiment.