Cell and Molecular Biology BIO 171 Laboratory

Name: Lerios, Ameer Muhmien J. Score:

Year/Section: BS BIO 3A Date Submitted:

ACTIVITY NO. 10

WESTERN BLOT: TESTING PROTEIN EXPRESSION LEVELS

Western blot is often used in research to separate and identify proteins. In this technique
a mixture of proteins is separated based on molecular weight, and thus by type, through gel
electrophoresis. These results are then transferred to a membrane producing a band for each
protein. The membrane is then incubated with labels antibodies specific to the protein of
interest.

The unbound antibody is washed off leaving only the bound antibody to the protein of
interest. The bound antibodies are then detected by developing the film. As the antibodies only
bind to the protein of interest, only one band should be visible. The thickness of the band
corresponds to the amount of protein present; thus doing a standard can indicate the amount of
protein present.

Objectives:
This activity aims to determine the expression of a particular protein in human cells.
Specifically, to visualize the expression levels of the macrophage co-receptor CCR5 in CRISPR-
Cas9 edited cell lines.

Virtual lab website:

https://www.labxchange.org/library/items/lb:LabXchange:ee936a17:lx_simulation:1

Protocol:
1. Create the Western blot "sandwich".
2. Transfer protein.
3. Block the membrane.
4. Incubate with primary antibody.
5. Wash the membrane.
6. Incubate with secondary antibody.
7. Wash the membrane.
8. Image the membrane.

Questions:
1. What is the principles of western blot?

Western blotting (also known as protein blotting or immunoblotting) is a sensitive and

quick method for detecting and characterizing proteins. It works on the basis of
immunochromatography, which separates proteins into polyacrylamide gels based on their
molecular weight. The separated proteins are subsequently transferred or electrotransferred
onto nitrocellulose membranes, where they are identified using specific primary antibodies and
secondary enzyme labeled antibodies and substrates.

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2. What is the purpose of TBST buffer?

TBST (or TTBS) (also known as Tween 20). It's a buffer that's used to wash
nitrocellulose membranes and microtiter plate wells in ELISA experiments. The main purpose of
TBST is in the blocking buffer and subsequent steps is to prevent none specific binding of
proteins. TBS blocking buffers are also the best choice for using phospo-specific antibodies to
identify phosphorylated protein molecules. The primary antibody will bind not only to the
phosphate on the target protein, but also to the phosphate in the PBS buffer, considerably
decreasing your target signal.

3. What is the function of nitrocellulose membrane?

Nitrocellulose is use for its fast binding and steady support. Because of its strong
protein-binding affinity, compatibility with a range of detection modalities (chemiluminescence,
chromogenic, and fluorescence), and capacity to immobilize proteins, glycoproteins, and nucleic
acids, nitrocellulose membranes are a popular matrix used in protein blotting.

4. What is the purpose of adding primary and secondary antibody?

The primary antibody recognizes the antigen in the sample, but the secondary antibody
can be engineered to include a fluorophore or enzyme complex for visualization. When two
antibodies are used to view an antigen, the color that will indicate the antigen's location can be
chosen.

5. Why there is a need to use shaker in the process?

A patchy and uneven areas on the blot is caused by the improper transfer of solution. If
air bubbles are caught between the gel and the membrane, the film will seem darker. It's also
crucial to utilize a shaker for entire incubation so that there's no uneven agitation.

6. What is the function of milk buffer?

Antibodies are proteins, therefore they may attach to the membrane in an indiscriminate
manner, to any site in the membrane. So, the function of milk includes many proteins that bind
to the membrane to decrease non-specific binding. Proteins in milk, on the other hand, bind to
the membrane in a specialized manner and thereby fill many sites.

7. Compare western blot to southern blot.

E.M. Southern discovered Southern blotting in 1975. Southern analysis or blotting is a
method for detecting a gene of interest in a DNA sample. Stages in this type of blotting
technique includes, Isolation of DNA, separation by electrophoresis, transfer to a suitable
medium, hybridization to probes, and visualization of the gene, if present. Southern blot is
commonly used in phylogenetic analysis and identification of gene arrangements. Meanwhile,
Western blotting, is used to detect proteins by isolating the proteins from a particular sample
and adding antibodies which is responsible for recognizing a specific amino-acid sequence. The
distinction is in the visualizing technique. Western blotting can be used to detect HIV antigens or
Hepatitis B surface antigens in blood. Another comparison between the two are, Primary and
secondary antibodies are used in Western blotting, whereas a radiolabeled (fluorescent) probe
or dye that binds to the DNA is used in Southern blotting.

8. What are the advantages and disadvantages of using western blot?

One of the advantages of using western blot is that it can theoretically serve as an
excellent early diagnostic tool, sensing even the tiniest immunogenic response from a virus or
bacteria in a patient sample, due to its ability to detect as low as 0.1 nanograms of protein in a
sample. The ability of the secondary antibody to boost the strength of the signal captured by the
imaging equipment adds to the sensitivity of an indirect western blot. Because of the increased

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Cell and Molecular Biology BIO 171 Laboratory

sensitivity, fewer antibodies are required for testing, resulting in significant cost savings in the
laboratory. Another advantage of western blot technique is its specificity when it comes to its
results. Gel electrophoresis divides a sample into proteins with varying sizes, charges, and
conformations. This procedure is a significant step toward detection in and of itself, as the
bands generated on the gel already provide information about the size of the protein or
polypeptide of interest. Secondly the specificity of the antibody-antigen interaction. Because
specialized antibodies have a high affinity for specific proteins, the technique can detect a target
protein in a mixture of 300,000 proteins.

Despite its sensitivity and specificity, a western blot can still produce erroneous results.
The disadvantage is that when an antibody reacts with a non-intended protein, as when a
patient being tested for HIV has tuberculosis or several parasite illnesses, a false-positive result
may occur. A false-negative result might readily occur if bigger proteins are not given enough
time to adequately move to the membrane. Improper blotting and processing frequently result in
skewed, fading, or even numerous bands, leaving test results open to the technician's
judgment. Another disadvantage is that it is high Cost and high technical demand. The cost of a
western blot is made up of several big individual expenses such as labeled antibodies, qualified
analyzers, and lab equipment. Western blotting is a complex technique that necessitates
precision in each stage for accurate identification of a sample's elements. A slight reagent
concentration or incubation period miscalculation can have severe consequences for the entire
operation. Finally, detection and imaging equipment such as chemiluminescent, fluorescent,
radioactive, or laser detection systems might be too expensive for the common microbiology
lab.

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9. What is the result of your simulation in this activity? Provide screenshots.

The ladder in the first lane is used to estimate the size of any detected proteins. During
the incubation with the primary antibody, you added a control antibody against alpha tubulin (55
kDa) that ensures that protein transfer from gel to membrane worked in principle and that you
had enough protein to be detected in each sample. We therefore expect to see the control
protein tubulin to be present in all samples at the same size of 55 kDa.

In contrast, we only expect to see the protein produced by CCR5 in the wild type macrophage
sample where this gene has not been disrupted by CRISPR-Cas9. The CCR5 protein is about
40 kDa in size. CCR5 should be absent in the pancreas cell culture since it is not expressed by
these cells. The cell lines edit 4 and edit 7 should also not contain any CCR5 protein if the
CRISPR-Cas9 gene disruption procedure worked in an ideal manner.

Predicted Results

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Actual Results

Ideal Results

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10. Your results show a faint band of CCR5 in the Edit 7 lane that is missing in the ideal
results. How can you explain this?
The gRNA that was used to edit cell line Edit 7 did not facilitate efficient CCR5 gene
disruption. As a result, Edit 7 expresses still low levels of CCR5

11. You need to select one CRISPR-edited cell line to continue with your experiments.
Predict which cell line (Edit 4 or Edit 7) will display a higher rate of HIV resistance in
your next and last experiment.

It is predicted that is cell line, edit 4 and the reason is due to successful deletion of CCR5.

12. Western blotting is a technique used to detect a:

A western blot is a method that is used in the laboratory for detection of specific protein
molecules from among a mixture of proteins. The said mixture can include all of the proteins
associated with a particular tissue or cell type. And also Western blots can be used to evaluate
the size of a protein that we are interested in, and for measurement of the amount of protein
expression.

13. Prior to Western blotting, proteins in the samples were separated using the following
technique:
Western blot is often used in research to separate and identify proteins. In this technique a
mixture of proteins is separated based on molecular weight, and thus by type, through gel
electrophoresis. These results are then transferred to a membrane producing a band for each
protein. We already have the knowledge in previous activity that gel electrophoresis is a
technique in which molecules that are charged, this includes the protein or DNA, are separated
according to physical properties as they are forced through a gel by an electrical current.
Commonly, these proteins are being separated using polyacrylamide gel electrophoresis
(PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins
within a single sample. In combination with western blotting, PAGE is a great analytical tool
when it comes to giving information on the proteins’ mass, charge, purity or presence. Several
forms of PAGE exist and can offer different types of information about the protein/s of interest.

14. In which order should the following steps involved in a Western blot test be
performed? 1 - visualize protein fluorescence; 2 - transfer proteins from gel onto a
membrane; 3 - incubate membrane with specific antibody
The correct order involved in performing Western blot test is 2, 3, 1 or transfer proteins from
gel onto a membrane, incubate membrane with specific antibody, and visualize protein
fluorescence.

15. Why does the nitrocellulose membrane have to be incubated with a 5% milk solution?
According from the virtual simulation conducted, the nitrocellulose membrane has to be
incubated with a 5% milk solution in order to block the membrane before antibody incubation

16. Which step creates the specificity towards the protein of interest?
Incubation with primary antibody

17. Provide a recorded video of the simulation of this activity. Include step-by-step
demonstrations.

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