Target Protein Identification and quantification
Target protein identification via SDS-PAGE and Western
Blot techniques
Skyler Gilreath, Andres Quintana
Results
SDS-PAGE
The data for part 1 of the two-part experiment included a gel that was created using the
Bio-Rad SDS-PAGE technique (Fig. 1A) and a standard curve graph that was created using the
values from the ladder key (Fig. 1B). The gel that was run, was then compared to the ladder key
provided by Bio-Rad which allowed for identification of the unknown protein samples via their
molecular weight.
A gel with 10 wells was used with lanes 1-5 being replicated to allow one gel to be used
for protein transfer for Part 2 and one gel for Coomassie staining. Once the run of the gel was
completed, it was divided in half and the first half was stained with Coomassie blue which
allowed for visualization of the bands (Fig. 1A). The instructor provided Coomassie stain gel
image showed 10 bands with each one representing a different molecular weight. The distance
values were measured for each band and a graph was created by plotting those distances vs the
log10 of the molecular weight (kDa). By doing this, determination of the molecular weight of the
unknown protein samples that were also run, as shown in Table 1, was completed. The standard
curve equation provided by Excel, Y=-25.852X+76.502, was used, where X is the log10 of the
molecular weights which we can now calculate, and Y would be the measured distance of band
migration. The molecular weight of the unknown samples was converted to correct values by
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Target Protein Identification and quantification
raising 10 to the power of the X. For unknown 1 the calculated molecular weight was 151.96
kDa, for unknown 2 the calculated molecular weight was 32.26 kDa, for the cell lysate; 23.62
kDa, and finally the tissue homogenate; 13.24 kDa. Compared to the theoretical molecular
weight of cytochrome C, 12.00 kDa, the results showed that the tissue homogenate contained the
most, due to its similar molecular weight, 13.24 kDa.
Table 1: SDS-PAGE gel calculations/results
Lane Number Sample Sample Composition Running Length
(cm)
Molecular Weight
(kDa)
1 Protein Ladder - 5.2 -
2 Unknown 1 20 μL 2.01 151.96
3 Unknown 2 10 μL unknown 2: 10 μL
H2O3.75 32.26
4 Cell Lysate 10 μL cell lysate: 10 μL
diH2O4.10 23.62
5 Tissue Homogenate 10 μL tissue homogenate:
10 μL diH2O4.75 13.24
Western Blot
Part two of the procedure, the Western Blot, was conducted for imaging of the unknown
proteins utilizing the Bio-Rad SDS-PAGE gel that was saved from Part one and transferring that
to the hydrophobic PVDF membrane. The results of the Western Blot were analyzed to
determine the presence of a specific protein, cytochrome c.
The imaging of the Western Blot revealed positive test results in lanes 2,4, and 5. This
confirmed the presence of the primary antibody that was being tested for, cytochrome C (Fig.
1C). This means that Unknown one was able to bind to the cytochrome c, and there were varying
amounts of this antibody in the Cell Lysate and Tissue Homogenate. This imaging as well as the
molecular weight calculations and professor provided results, indicated that unknown one is
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Target Protein Identification and quantification
cytochrome c and there is a greater amount of cytochrome c in the Tissue Homogenate than the
Cell Lysate.
Materials and Methods
SDS-PAGE- Materials
All of the materials that were utilized during the SDS-PAGE procedure include: Bio-Rad
Mini-Protean electrophoresis chamber (Catalog #1658005); Bio-Rad Turbo Transfer system
(Catalog #17001917); Bio-Rad pre-cast, 4-20%, polyacrylamide gels with 10 wells (Catalog
#4561094); Mixture of 2X Laemmli’s sample buffer containing ß-mercaptoethanol; Bio-Rad
Coomassie stain (Catalog #1610786); Bio-Rad 1X Tris/Glycine/SDS running buffer (Catalog #
1610732); Bio-Rad 1X turbo transfer buffer (part of system); Bio-Rad PVDF membrane
(Catalog #1704156); Bio-Rad transfer stacks/filter paper (part of system); Bio-Rad Precision
Plus protein ladder (Catalog #1610374); Methanol (MeOH); protein samples: Unknown #1 (3.0
μg), Unknown #2 (1.5 μg), Cell Lysate (2.0 μg), Tissue Homogenate (2.0 μg); Deionized water.
SDS-PAGE- Methods
Preparing Protein Samples
A dilution of unknown protein #1 was made using diH2O as a dilutant to create a final
volume of 20 μL. A dilution of unknown protein #2 containing 1.5 μg of protein was created
using the same dilutant. Two separate dilutions of the cell lysate and tissue homogenate from
previous experiments were used containing 2.0 μg each, once again with the same dilution and
final volume. Each protein sample had 20 μL of the mixture of 2X Laemmli’s sample buffer and
were all boiled for three minutes at 100°C. All of the samples were then briefly spun in a mini
centrifuge and were loaded into the gel wells. The gel electrophoresis chamber was assembled
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Target Protein Identification and quantification
under the supervision of the lab TA and each protein sample was carefully loaded, as well as the
protein ladder.
Gel Electrophoresis
The chamber was filled to the appropriate line with 1X running buffer and the chamber
lid was connected to the post matching respectively, red to red and black to black. The electrode
leads were inserted to the power supply and the chamber was programmed to perform
electrophoresis at 300 V for 10 to 15 minutes. Once the run was complete the power supply was
disconnected, and the gel was removed. The gel was removed from the plate and was divided in
half with a razor blade, one for protein transfer and the other for the Coomassie stain.
Turbo Transfer
A single PVDF membrane was soaked in 100% methanol and transferred to a 1X turbo
transfer buffer, while 2 turbo transfer stacks were soaking in 1X turbo transfer buffer for
approximately 5 minutes. Forceps were used to prevent decontamination while transferring the
PVDF membrane onto one of the transfer stacks. The gel was placed onto the PVDF membrane
using forceps and the second turbo transfer stack was placed on top of the gel creating a
“sandwich”. Before transferring to a transfer plate, a gel roller was used to remove any air
bubbles so as not to interfere with transfer of proteins. The turbo transfer apparatus was
programmed to perform the transfer and once it was complete the PVDF membrane was
removed and stored in a container for further analysis.
Staining the Gel to Calculate Molecular Weight of Unknown Protein Samples
The other half of the gel was transferred to a container filled with tap water and washed
for 5 minutes on a rocker to remove the SDS from the gel. The water was carefully poured into
the sink and this procedure was repeated two more times. The Bio-Safe Coomassie stain was
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Target Protein Identification and quantification
added to the container until it completely covered the gel and was incubated in the stain. The rest
of the procedure which includes the de-staining of the gel and usage of the gel imager was
conducted by another lab group and the results of that were provided by the professor. The
distances were recorded from the provided image and the data was used to graph a standard
curve.
Western Blot- Materials
All of the materials that were utilized during the Western Blot procedure include: Stored
PVDF membrane; Methanol (MeOH); TBS-T wash buffer (Tris-buffered saline with 0.05% v/v
Tween-20); Blocking solution (2.0% w/v non-fat dry milk in TBS-T); BD Pharmingen primary
antibody, purified mouse anti-cytochrome c, diluted 1:200 in blocking solution (Catalog
#556433); Bio-Rad secondary antibody, goat anti-mouse HRP conjugate, diluted 1:3000 in
blocking solution (Catalog #170-5047); Bio-Rad Clarity Western ECL Blotting Substrates
(Catalog #1705060S); and finally a Bio-Rad ChemiDoc imager.
Western Blot- Methods
A dried PVDF membrane was soaked in 100 % methanol and washed in TBS-T buffer for
three minutes. The TBS-T was poured gently into the sink and the membrane was incubated in
blocking solution for approximately 30 minutes, the blocking solution was then discarded and
then the membrane was incubated in the primary antibody for one hour. The antibody was
discarded into the sink and the PVDF membrane was washed three times for 5 minutes each in
TBS-T. The PVDF membrane was then incubated in the secondary antibody for one hour,
antibody was discarded, and once again the PVDF membrane was washed three times for 5
minutes each in TBS-T. The two ECL solutions were mixed in a one-to-one ratio and the
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Target Protein Identification and quantification
membrane was incubated in this mixture for 5 minutes in a drawer without light and immediately
transferred to the Bio-Rad ChemiDoc imager which was operated by the lab professor.
References
1. DeWitt, D. A., Sellers, J.A., Solitro, A. R., Townsend, B. E, Winter J. N. (2024)
Exploring Cell Biology Laboratory Manual, Ninth Edition, Liberty University.
Lynchburg, VA.
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Target Protein Identification and quantification
Figure 1. Identifying Cytochrome C via SDS-PAGE, Gel Staining, and Western Blot. (A)
The Bio-Rad SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) technique
was used to vertically separate the unknown proteins by molecular weight which was made
visible through Coomassie staining. In order, lanes 1-5 contain: standard protein ladder,
Unknown 1 (3.0 μg), Unknown 2 (1.5 μg), Cell lysate (2.0 μg), and Tissue homogenate (2.0 μg).
(B) The values obtained from the standard ladder key (log10) and the distance travelled measured
from the stain (mm), were used to create a standard curve graph. Once the scatter plot was
created a trend line was added and the equation was used to calculate the unknown proteins
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AC
B
Target Protein Identification and quantification
molecular weights (kDa). For unknown 1 the calculated molecular weight was 151.96 kDa, for
unknown 2 the calculated molecular weight was 32.26 kDa, for the cell lysate; 23.62 kDa, and
finally the tissue homogenate; 13.24 kDa. (C) A Western Blot was used to visualize the samples
and indicated that the Tissue Homogenate contained the most cytochrome c due to the similar
molecular weights. In order, lanes 1-5 contain: standard protein ladder, Unknown 1 (3.0 μg),
Unknown 2 (1.5 μg), Cell lysate (2.0 μg), and Tissue homogenate (2.0 μg).
Figure 2. Fold Difference of Cytochrome C in Cell Lysate vs Tissue Homogenate indicates
no Statistical Significance. (A) Values provided by the western quantitative analysis data were
averaged and plotted in a column graph. This showed that there was 1.81x more cytochrome C in
the tissue homogenate (beef liver) compared to the cell lysate (rat2 fibroblast). Standard
deviation for both averages were calculated and added to the graph as error bars. Furthermore, at
T-Test was conducted on the data which provided a P value of 0.3495 which is greater than .05,
the value needed in order to be considered statistically significant. This means that there is no
biological difference, and the change is most likely due to chance.
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