Target protein identification and quantification
Target protein identification and quantification via SDS-
PAGE, gel staining, and Western Blot techniques
Name 1, Name 2
From the Department of Biology and Chemistry, Liberty University
Results
SDS-PAGE
The data from day 1 includes a gel made using the SDS-PAGE technique along with a
corresponding graph (Fig. 1A)(Fig. 1C). The gel was compared to the Dual Color Precision Plus
Protein Standards Ladder key provided by BioRad ultimately allowing for the unknown protein
samples to be identified based on their molecular weight (Fig. 1B).
After the gel ran, it was stained with Coomassie Blue to allow for visualization of the
bands (Fig. 1A). Comparison of the ladder key to the sample protein ladder in lane one showed a
match for the first nine bands while the tenth (representing 10 kD) was absent on the
experimental sample. The values from the ladder key were then used to create the standard curve
graph by plotting the distance traveled (mm) versus the log10 of the molecular weights (kDa)
(Fig. 1C). Based on the results of this graph, it was possible to determine the molecular weight of
each unknown protein sample as shown in Table 1. This was done by using the trendline
equation for the graph, y = (-17.7)(x) + 46.5, wherein “y” is the measured running length of each
sample, thus allowing for the log10 of the molecular weights “x” to be calculated. From there we
were able to convert the log values to normal values by raising 10 to the power of the log values,
finally giving us the molecular weights of each unknown protein sample. The average molecular
weight of Unknown 1 was 373.30 kDa and Unknown 2 was 359.01 kDa.
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Target protein identification and quantification
Table 1: SDS-PAGE gel results.
Lane Number Sample Sample Composition Running Length Molecular Weight
1 Protein Ladder - 3.1 cm -
2 Unknown 1: 3.0 μg 20 μL Unknown 1 1.05 cm 369.66 kDa
3 Unknown 1: 1.5 μg 10 μL Unknown 1;
10 μL H20
0.9 cm 376.94 kDa
4 Unknown 2: 2.0 μg 20 μL Unknown 2 1.35 cm 355.51 kDa
5 Unknown 2: 1.0 μg 10 μL Unknown 2;
10 μL H20
1.2 cm 362.51 kDa
Western Blot
The Western Blot procedure allows for additional imaging of the unknown protein
mixtures by transferring the gel from the SDS-PAGE to a hydrophobic PVDF membrane (Fig.
1D). This was used to determine both the presence of a protein in addition to the relative quantity
of the proteins.
Imaging of the Western Blot produced after the gel electrophoresis revealed distinct
bands indicating a positive test result in both lanes 2 and 3, and the absence of bands indicating a
negative test result in lanes 4 and 5 (Fig. 1D). As lanes 2 and 3 contained varying concentrations
of Unknown protein 1, the development of bands confirms the presence of Unknown 1 on the
Western Blot. It also indicates that the primary antibody, purified mouse anti-cytochrome c, was
able to bond to protein 1. On the other hand, the lack of bands in lanes 4 and 5 where Unknown
protein 2 was loaded indicated that Unknown 2 was not present on the Western blot, and
furthermore that the primary antibody was unable to bind to protein 2. These results, along with
the molecular weights determined by the SDS page, allowed for the identification of Unknown 1
as cytochrome c. The results were not clear enough to quantify the present proteins.
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Target protein identification and quantification
Materials and Methods
Part 1 - Materials and Equipment
The following is a complete list of materials used in Part 1: Bio-Rad Mini-Protean
electrophoresis chamber (Catalog #1658005); Bio-Rad Turbo Transfer System (Catalog
#17001917); Bio-Rad precast, 4-20%, polyacrylamide gels with 10 wells (Catalog #4561094),
Mixture of 2X Laemmli's sample buffer containing β-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 (0.15 ug/uL) and
Unknown 2 (0.10 ug/uL); Deionized water.
Part 1 - Methods
Part A: Preparing Protein Samples
Two protein dilutions of Unknown 1 were prepared, one containing 3.0 μg of protein and
the other containing 1.5 μg of protein, using diH2O as the diluent and final volumes of 20 μL
each. Next, two protein dilutions of Unknown 2 were prepared, one containing 2.0 μg of protein
and the other containing 1.0 μg of protein, using diH2O as the diluent and final volumes of 20
μL each. Once diluted, 20 μL of the 2X Laemmli's sample buffer and 10X β-mercaptoethanol
mixture was added to each protein sample. Then, all four samples were boiled for 3 min at 100°C
using the heat block and briefly spun in a mini centrifuge to collect the tube contents in the
bottom of the tube. The gel electrophoresis chamber was assembled as described in the Bio-Rad
SDS-PAGE video and gel-loading micropipette tips were used to load the protein ladder and
protein samples into the lanes of the gel.
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Target protein identification and quantification
Part B: Gel Electrophoresis
The electrophoresis chamber was filled to the appropriate line with 1X running buffer,
the chamber lid was connected to the posts (matching red to red and black to black) and the
electrode leads were connected to the power supply (matching red to red and black to black). The
power supply was programmed to perform electrophoresis at 300 V for 10-15 minutes and
electrophoresis was begun. Once the run was complete, the power supply was turned off and
electrode leads were disconnected before removing the lid from the electrophoresis chamber.
The two gel plates were separated and while the gel was still on the gel plate, it was divided in
half with a razor blade. One half was used in Part C for transferring the proteins in preparation
for the Western Blot while the second half was used in Part D for staining the gel.
Part C: Turbo Transfer
To begin, a single PVDF membrane was soaked in methanol for 3 minutes while two
Turbo Transfer stacks were soaked in 1X Turbo Transfer buffer for 5 min. Next, the PVDF
membrane was transferred onto one of the transfer stacks and the gel plate was flipped so the
protein side of the gel was facing down. Half of the gel from the gel plate in Part B was
transferred onto the PVDF membrane and the second Turbo Transfer was stacked on top of the
gel, making a "sandwich". A gel roller was used to roll out any air bubbles before the entire
sandwich was placed in a transfer plate, locking the lid in place. The Turbo Transfer apparatus
was programmed to perform an appropriate transfer and run. Once the transfer was complete, the
PVDF membrane was removed and stored in a labeled plastic container at room temperature
until Part 2.
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Target protein identification and quantification
Part D: Staining the Gel to Calculate the Molecular Weight of Unknown Proteins
The second half of the gel from Part B was transferred into a plastic container filled with
tap water and washed for 5 minutes with agitation on a rocker. The tap water was drained and
this step was repeated two more times. After the third tap water rinse, Coomassie stain was
added to the container until the gel was covered completely and it was then incubated overnight
(at least) with gentle agitation.
The following day, the used Coomassie stain was discarded and the gel was washed in
tap water for 20 minutes with gentle agitation on a rocker. The gel was then imaged and the
distances traveled for each protein band in the protein ladder along with each unknown sample
were measured and recorded. A standard curve graph was made using the distance traveled (mm)
versus the logarithm10 of the molecular weight (kDa). The equation of the standard curve line
was then used to determine the molecular weights of each of the unknown protein samples.
Part 2 - Materials and Equipment
The following is a complete list of materials used in Part 2: Stored PVDF membrane
from Part 1C; 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 1:3,000 in
blocking solution (Catalog #170-5047); Bio-Rad Clarity Western ECL Blotting Substrates
(Catalog #1705060S); Bio-Rad ChemiDoc Imager.
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Target protein identification and quantification
Part 2 - Methods
The stored PVDF membrane from Part 1C was soaked in methanol for 3 minutes then
transferred to the TBS-T wash buffer and washed for 3 min while rocking. TBS-T was discarded
and the PVDF membrane was incubated in a blocking solution, rocking for 30 minutes at room
temperature. The blocking solution was discarded and the PVDF membrane was incubated in
500 mL of primary antibody, keeping the membrane stationary for 1 hour at room temperature.
The primary antibody was discarded and the PVDF membrane was washed in TBS-T while
rocking 3 times for 5 minutes each. The PVDF membrane was then incubated in a secondary
antibody, rocking for 1 hour at room temperature. The secondary antibody was discarded and the
PVDF membrane was once again washed in TBS-T while rocking 3 times for 5 minutes. Two
ECL solutions in a 1:1 ratio with a final volume of 2 mL were then mixed and the PVDF
membrane was incubated in the ECL mixture for 5 minutes in the dark. A Bio-Rad ChemiDoc
imager was used to take a picture of the Western blot.
References
1. DeWitt, D. A., Sellers, J.A., Solitro, A. R., Townsend, B. E. (2020) Exploring Cell Biology
Laboratory Manual, Eighth Edition, Liberty University. Lynchburg, VA.
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Target protein identification and quantification
Figure 1. Identifying Cytochrome C using SDS-PAGE, Gel Staining, and Western Blot
Techniques. (A) The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
technique was used to separate proteins vertically based on their molecular weights after staining
with Coomassie dye. From left to right, the contents of lanes 1-5 are as follows: protein ladder,
Unknown 1 (3.0 μg), Unknown 1 (1.5 μg), Unknown 2 (2.0 μg), and Unknown 2 (1.0 μg).
(B) The Dual Color Precision Plus Protein Standards Ladder key provided by BioRad was used
as a reference for determining the molecular weight of each band. (C) The values from the ladder
key were then used to create the standard curve graph by plotting the distance traveled (mm)
versus the log10 of the molecular weights (kDa). From the graph, a trendline was plotted and was
then used to calculate the molecular weights of the unknown proteins. (D) After the results from
the gel and graph were interpreted, a Western Blot was then used to visualize all of the protein
samples collected. From left to right, the contents of lanes 1-5 are as follows: protein ladder,
Unknown 1 (3.0 μg), Unknown 1 (1.5 μg), Unknown 2 (2.0 μg), and Unknown 2 (1.0 μg).
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