Characterization of Proteins: Protein Gels, Western Blotting and Immunodetection

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BIO 101B – MOLECULES, GENES AND CELLS
LABORATORY #4:
CHARACTERIZATION OF PROTEINS:
PROTEIN GELS, WESTERN BLOTTING,
AND IMMUNODETECTION
October 25, 2017
LABORATORY #4: CHARACTERIZATION OF PROTEINS: PROTEIN GELS, WESTERN BLOTTING, AND IMMUNODETECTION OCTOBER 25, 2017
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DAY ONE: Preparation of Sample Cell Lysates
Objective:
The object of this experiment was to determine the size of proteins extracts from the heart tissue
of a mouse by running a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and performing a western blot.
40  80  5 
Results:
At the end of the experiment, we could determine the size of
the beta-actin protein by using the immunodetection. The
beta-actin protein was about 50 kDa which is exactly what
we were expecting. In the Coomassie Blue stained gel,
visible in Figure 1, shows the protein bands on the protein
ladder. Figure 2 shows the immuno-stained blotted
membranes. The blots were stained using an anti-beta-actin
antibody primary antibody and Alkaline Phosphatase as a
secondary antibody. The blots show a labeled band at
50kDa however, I am not
80  5 
sure if the intensity of the
stained band correlates
with the amount of protein
loaded into the well. The
blot in Figure 2 has no
background staining and
the only visible band is in
Figure 1: The Coomassie Blue stained
gel. The Leftmost lane contains 40  of
cell lysate while the middle lane contains
80  of cell lysate and the right lane the
5  protein ladder or marker. The
numbers in the picture show the size of
proteins in kilodalton (kDa).
the 80  lane. I worry that
there was an error with our
experiment that resulted in the lack of background staining. In
Figure 1 there is a clear difference in the intensity of the lanes
depending on the concentration of the cell lysate but that does
not seem to transfer over to the blot.
Figure 2: shows the beta actin in the red
square next to the 5  protein ladder. The
ladder is labeled with the size of protein
bands. We concluded that the protein in
the cell lysate was about 50 kDa.
LABORATORY #4: CHARACTERIZATION OF PROTEINS: PROTEIN GELS, WESTERN BLOTTING, AND IMMUNODETECTION OCTOBER 25, 2017
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Conclusion:
Our results indicated that the beta-actin protein was detected. The Coomassie Blue stained gel
showed good separation of proteins by SDS-PAGE and the expected sized of the detected
band matched what we found on the staining as well (Approximately 50 kDa). We only found a
single labeled band which indicates high specificity of the staining and antibody.
This discovery of high specificity in the stain and antibody is good because assuring the integrity
and validity of the samples as well as the specificity of the antibody tends to be a problem with
immunoblotting. As important and critical as the Western Blotting technique has been to
research over the last 35 years, there are still several limitations with the process. For example,
not all experiments are as effective as ours. Some Western Blot experiments are being
conducted and reported without the requisite controls (McDonough et al. 2015). A study by
Gilda et al. 2015, compared ubiquitinated proteins in rat heart and liver samples and showed
the different results depending on the antibody utilized. The antibodies. The study concluded
that the antibodies differ in their ability to detect ubiquitinated proteins.
In our experiment we used the Alkaline Phosphatase antibody which is a goat anti mouse beta
actin antibody. The anti-body is generated in the blood of a goat against mouse beta-actin
protein. Although our experiment matched the specificity of the antibodies well, other
experiments must consider integrity, validity and specificity when designing an experiment.
LABORATORY #4: CHARACTERIZATION OF PROTEINS: PROTEIN GELS, WESTERN BLOTTING, AND IMMUNODETECTION OCTOBER 25, 2017
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BIBLOGRAPHY
Gilda, Jennifer E., et al. “Western Blotting Inaccuracies with Unverified Antibodies: Need for a
Western Blotting Minimal Reporting Standard (WBMRS).” PLOS ONE, Public Library of
Science, 19 Aug. 2015, doi.org/10.1371/journal.pone.0135392.
A McDonough, Alicia & Veiras, Luciana & Minas, Jacqueline & Lee Ralph, Donna. (2014).
Considerations when quantitating protein abundance by immunoblot. American journal
of physiology. Cell physiology. 308. ajpcell.00400.2014. 10.1152/ajpcell.00400.2014.
LABORATORY #4: CHARACTERIZATION OF PROTEINS: PROTEIN GELS, WESTERN BLOTTING, AND IMMUNODETECTION OCTOBER 25, 2017
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