Carlina Vanjo

Dr. Phillip Funk

Immunobiology Lab
26 February 2015
Western Blotting
Introduction
Western blots are very useful when trying to qualitatively determine
the molecular weight of proteins in addition to their reactivity towards
antibodies. It was originally hypothesized that cells exposed to an antigen
(that binds to their surface antigen receptor) leads to an increase in bcl-xL
expression, therefore protecting the cells as they fight against the antigen. In
order to test this hypothesis, a western blot was performed using the cell
lysates produced and quantitated during the first part of this experiment. For
this experiment, Dr. Funks cell lysates were used because the cell lysates
previously created were unusable.
Agarose gel electrophoresis separates the proteins by their molecular
weight, detecting the presence and amount of the protein bcl-xL. However,
antibodies cannot be added directly into the gel because they will only
migrate slowly through the gel and then bind with their antigen. Because of
this, a membrane composed of nitrocellulose is placed on top of the gel so
that the proteins can be transferred to it with the help of an electric current.
Once the proteins have transferred onto the membrane, the antibodies can
be added. By soaking the membrane in albumin, all sites on the membrane

that do not already contain proteins are blocked, ensuring the antibodies
bind to their CDR loops and not the membrane itself. A primary antibody,
rabbit bcl-xL, is then added to act against bcl-xL. This is to detect the
antibody of interest. Once the primary antibody is added to the membrane, a
secondary antibody (goat anti-rabbit bcl-xL) bound to an enzyme (alkaline
phosphatase) is added to counteract the constant regions of the primary
antibody. A substrate is then added so that when it is cleaved by alkaline
phosphatase, a colored product appears and signifies the binding of the
primary antibody to the bcl-xL protein in the cell lysates. By observing this
colored product, the cell lysates expressing bcl-xL become easily
determinable.

Methods
Four clean microcentrifuge tubes were obtained and labeled. In each
tube, 25g of loading dye was added to 25g of one cell lysate. The four
tubes were then boiled in a heat block for five minutes and cooled in an ice
bath for two minutes. Once the samples were taken out of the ice bath, they
were centrifuged for a few seconds and loaded into the agarose gel. A
marker containing 5L of prestained molecular weight standards was
provided and loaded into lane 1 of the gel. Lane 2 contained sample A
(negative control cell lysate), lane 3 contained sample B (experimental cell
lysate), lane 4 contained sample C (experimental cell lysate), and lane 5

contained sample D (positive control cell lysate). The voltage was then set to
200V and the gel underwent electrophoresis for 45 minutes.
Once the electrophoresis was complete, the gel was removed and
placed into a dish containing transfer buffer with the transfer cassette. While
still submerged in the transfer buffer, the first fiber pad was wet with buffer
and placed on the cassette. A buffer-soaked piece of filter paper was then
placed on top of the fiber pad. A pipet was rolled over the filter paper to
remove any bubbles. The gel was the positioned on top of the filter paper,
and again a pipet was used to remove any bubbles. Then a sheet of
nitrocellulose was carefully laid on top of the gel so as to exactly cover its
surface. Any bubbles were once again removed with a pipet. Another piece of
filter paper was placed on top of the nitrocellulose, followed by another fiber
pad, and then the cassette was closed and locked. The transfer cassette was
then positioned into the transfer apparatus, filled with buffer, and sealed
with a lid. The power current was set to 100mA and run for one hour. Once
the transfer was complete, the cassette was unlocked and the gel was
removed. The nitrocellulose membrane was also removed, placed in blocking
buffer, and marked in the corner with a razor blade to maintain proper
orientation. The fiber pads, transfer cassettes, and gel tanks were all washed
and put away.
A week later, the blot was placed in a dish with 5mL of wash solution
and swirled for five minutes. The wash was discarded and the blot was
washed again with 5mL of wash solution, swirled for five minutes, and again

the wash was discarded. The blot was then incubated with 10mL of primary
antibody solution on a rocker platform for 45 minutes. The primary antibody
solution was then poured back into its container and the blot was washed
twice with washing solution. The blot was then incubated with 10mL of the
secondary antibody solution for 45 minutes. Once this was complete, the
secondary antibody was returned to its container and the blot was washed
twice with washing solution. The blot was then incubated with 5mL of
substrate until colored bands were easily identifiable. The blot was then
transferred to water and the substrate solution was discarded.

Results
By comparing the Rf values of the standard proteins in the marker to
their known molecular weights, a formula can be generated so as to
calculate the molecular weights of the samples in lanes 3 and 5. Graphing
the Rf values versus the log of the molecular weights provides a linear graph,
whose formula can then be more easily used to solve for the molecular
weights of the proteins in lanes 3 and 5. The Rf values were found by dividing
the migrating distances of the proteins by the total distance of migration for
the dye font. The Rf values were used instead of the migrating distances
themselves because the data collected from the western blot is all relative.
The molecular weights of the samples were then compared with the
molecular weights of the standards in lanes 3 and 5. By substituting the

migrating distances for x, the molecular weights of the proteins in the

samples can be calculated by taking the inverse log of the y value. Using this
method, the molecular weight of the protein in lane 3 was calculated to be
111,846 Da and the molecular weight of the protein in lane 5 was calculated
to be 76,489 Da.
Table 1 shows the molecular weight values of the known standard
proteins in addition to their migrating distances and Rf values. The Rf values
were found by dividing the migrating distances of the proteins by the total
distance of migration for the dye font. The Rf values were used instead of the
migrating distances themselves because the data collected from the western
blot is largely relative. Once calculated, the molecular weights of the
samples in lanes 3 and 5 were added to the table so as to more easily
compare them with the molecular weights of the known samples.

Graphical Relationshoip of Rf Value and Molecular Weight of Standard Proteins

6
5

f(x) = - 1.83x + 5.34

R = 0.93

4
Log of Molecular Weight

3
2
1
0
0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Rf value

Figure 1: Graphical Relationship of Rf Values and Molecular Weights

of Standard Proteins. Figure 1 shows the Rf of the proteins plotted against
their molecular weights. The log function was used on the molecular weights
so as to form a more linear graph. The molecular weights of the proteins
found in lanes 3 and 5 were calculated substituting their migrating Rf values
into the equation for the trendline and taking the inverse log of the y value.
Table 1: Migrating Distances and Molecular Weights of Samples and
Standards.
Label
Distance
Molecular
Rf Value
Migrated (cm)
Weight (Da)
Myosin
0.3
199,356
0.05
S-Glucosidase
0.8
116,275
0.13
BSA
1.3
79,551
0.22
Ovalbumin
1.8
52,908
0.30
Carbonic
2.6
37,350
0.43
Anhydrase
Soybean trypsin
3.5
29,223
0.58
inhibitor
Lysozyme
3.8
19,834
0.63
Aprotinin
4.2
6,873
0.70
Sample B (lane 3)
1.0
111,846
0.16
Sample D (lane 5)
1.5
76,489
0.25

Figure 2: Western Blot. Figure 2 is a picture of the western blot obtained

from this experiment.
Discussion
Given the results of the experiment, the hypothesis does seem to be
supported. This is because the experimental cell lysate group exhibited a
band on the gel, signifying that the bcl-xL protein was present and being
expressed (lane 3). The positive control cell lysate also exhibited a band,
signifying that the bcl-xL protein was present and being expressed (lane 5).
The combination of these two events supports the idea that the data did not
occur due to chance.
By comparing the data of the samples and the standard proteins,
similarities of the values are easily seen. The migrating distances and R f
values of S-Glucosidase and BSA are very similar to that of the bands visible
in lanes 3 and 5.
In addition, graphing the Rf values of the standard proteins produced a
formula that was used to calculate the molecular weights of the proteins in

lanes 3 and 5. This formula describes the relationship between the Rf value
and molecular weight and is supported by the R2 value. The R2 value shows
that the formula can be used in confidence, as it states that the goodness of
fit is 0.92833 (roughly 93%). This value is high and ensures that the data
collected is of high quality. Since the western blot provides qualitative data,
more tests will have to be carried out in order to calculate the exact amount
of bcl-xL in lanes 3 and 5.