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Cell Biology Lab
Section 002
Tom Page
Lab Partners:Carson, Laina, Shannon
Using Western Blotting Techniques to Analyze Isolated BCL-2 Protein
Abstract: Western blotting is a common method that allows scientists to examine certain proteins
in a sample that are at a very low concentration. The experimental procedure involves the isolation
of the BCL-2 protein, treatment of the protein by SDS-PAGE, and western blotting the protein for
examination. The purpose of this technique is to observe the effects of different treatments in which
cell cultures were grown on the amount of BCL-2 protein produced by each culture. The four
treatments included 2% FBS, 5% FBS, 10% FBS, and 10% FBS + Camptothecin. The results
supported the hypothesis that the greater concentration of serum with no added chemicals would
yield the greatest concentration of BCL-2 protein. The thickest and most intense band of protein
from the gel electrophoresis was from the 10% FBS treatment (Figure 1). The BCL-2 apoptotic
pathway protein promotes cell survival, and therefore is found in the 10% serum which has the most
nutrients present.
Introduction: Cells produce certain proteins based on their responses to external and internal
factors. To analyze these proteins, many techniques can be used, but first the proteins that will be
examined must be isolated beforehand. The purpose of this lab is to become familiar with the
concepts and uses of protein isolation, SDS-PAGE, and Western Blotting. From these techniques,
the objective is to determine the levels of a protein known as BCL-2 present in cell cultures grown
at varying growth serum levels. BCL-2 is a protein that is involved in the apoptotic pathway which
prevents cell death (Dressler et. al, 1993). Fetal Bovine Serum (FBS) was added in varying
amounts to the different cell cultures. Flask A consisted of 2% FBS, Flask B 5%, Flask C 10%, and
Flask D 10% plus camptothecin. FBS is a serum that contains many nutrients and growth factors
that aid in cell development and proper cellular function and camptothecin is a chemical that is
potentially toxic to cells. The BCL-2 protein concentration should be the greatest in the 10% serum
with a higher concentration of FBS. Likewise, the BCL-2 concentration should be the lowest in
either the 2% serum or the serum containing the toxic chemical camptothecin.
Protein isolation using cells from the differing amounts of serum and camptothecin is the
first step in this technique. M-PER with protease and phosphatase inhibitors are added to each flask
to treat the cell cultures. M-PER, Mammalian Protein Extraction Reagent, is designed to provide
very efficient protein extraction from cultured cells (Irvine et. al, 1990). This reagent is a lysis
reagent that dissolves the cell’s plasma membrane and allows for the extraction of protein from the
intracellular area (Irvine et. al, 1990). Proteases are enzymes that break down the proteins into
smaller amino acid chains. After the cells are allowed to incubate in this environment, the cells
rupture and allow for proteins to be released. Furthermore, the protein then needs to be quantitated
and this is completed using the BCA protein assay. This technique uses the reduction of copper II
ions to copper I ions and combines this with the observation of spectrophotometric intensities of
color (Dressler et. al, 1993). The color change is directly proportional to the concentration of
protein in the sample. Therefore, the specific amounts of protein loaded in the gel will be the same
for all wells since the concentration is known. The BCA protein assay technique uses a working
reagent that is added to each of the wells, and this reagent has enzymes that form colors upon
reacting (Dressler et. al, 1993). The darker the color, the more protein present in the cell cultures.
After proteins have been isolated, SDS-polyacrylamide gel electrophoresis, or SDS-PAGE,
is conducted. This process is similar to DNA electrophoresis in that different proteins are separated
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through the gel medium by size using an electric field to attract them from one side of the gel to the
opposite. Instead of agarose gel for DNA, SDS-PAGE consists of an acrylamide gel. Proteins can
also have negative, positive, or neutral charges depending on their primary structure of amino acid
chains and are also able to fold upon themselves into shapes that may travel at different rates
through the gel (Tsuji et. al, 1989). Sodium Dodecyl Sulfate (SDS) is a detergent that eliminates
these two possible factors of affecting protein travel through the gel medium. SDS is present in
most of the reagents to keep the proteins denatured. SDS has a negative charge and therefore
denotes a negative charge on proteins that it binds to. This detergent also denatures proteins by
removing all secondary and tertiary structures so all proteins are basically the same shape in their
primary structure (Irvine et. al, 1990). The negatively charged proteins can then migrate toward the
positive end of the gel electrophoresis apparatus.
In western blotting, the protein samples are run on a polyacrylamide gel that is made up of
tunnels integrated through weaved fibers (Dressler et. al, 1993). The smaller-sized chains of
proteins will be able to maneuver themselves through the gel faster than the larger ones. This
results in the distinct separation of smaller and larger proteins while proteins of the same size create
distinct bands in the gel since they travel approximately the same distances. Since the BCL-2
proteins for these cell cultures may be present at very low concentrations, western blotting can be
used to identify the lesser amounts that have traveled through the polyacrylamide gel. After the
proteins have been run through the SDS-PAGE process, they need to be transferred to another
matrix known as nitrocellulose (Dressler et. al, 1993). They are transferred to the nitrocellulose by
forming a gel “sandwich” which can then have an electric current sent through it and easily transfer
an identical pattern of proteins onto the nitrocellulose.
After the transfer from the gel to nitrocellulose matrix where proteins then become trapped
in the pores, protein specific antibodies can be used to detect, in this case, the BCL-2 proteins.
Antibodies are proteins that bind to other proteins with a specific shape or configuration (Tsuji et.
al, 1989). An attached antibody resembles a Y with two arms composed of one heavy and one light
chain and a tail composed of two heavy chains (Tsuji et. al, 1989). The primary antibody used was
rabbit anti-BCL-2 antibody and this antibody bound directly to the BCL-2 proteins in the
nitrocellulose pores. Beforehand, 5% milk in TBST solution was used to fill in the remaining pores
so no other proteins or impurities could remain in the nitrocellulose pores. Also, the pores were
filled so antibodies did not bind them and give off light where no protein was present. Then, the
secondary antibody used was goat anti-rabbit antibody which had HRP bound to the end of it. HRP,
Horse Radish Peroxidase, can be detected colorimetrically through an enzymatic reaction and gives
off luminescence that can be seen as bands of protein on the nitrocellulose matrix (Tsuji et. al,
1989). The membrane was washed in TBST due to the high affinity of antibodies to bind to
proteins and there were excess antibodies that needed to be washed off. The amount of BCL-2
protein can then be examined by analyzing the intensity of light given off by each band on the
antibody-treated nitrocellulose matrix.
Materials and Methods: Part I: Obtain the cell cultures containing 2% serum, 5% serum, 10%
serum, and 10% serum plus camptothecin and remove the media from the cell cultures. Wash the
cells with PBS to remove any excess media or impurities. Add 100 µl of M-PER with protease and
phosphatase inhibitors to allow for easy protein extraction. Scrape the cells from the dish and
incubate on ice for 35 minutes to allow for rupturing and opening of cell membranes. Spin the
samples at 4oC for 10 minutes at 12,000 rpm. Move the supernatent to a new tube since it has the
proteins for experimentation.
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Conduct a BCA protein assay by pipetting 25 µl of each standard into the top 10 wells.
These standards range in concentration from 2 mg/mL all the way down to 25 µg/mL. Measure the
absorbance of the standards at 562 nm and compare it to samples from the experiment. Add 5 µl of
each of the four protein samples to specific wells and add 20 µl of M-PER lysis buffer to that. Add
200 µl of working reagent, which has enzymes that will react to form the specific color, to the
samples. Cover and incubate at 37oC for 30 minutes to find protein concentrations.
Part II: Obtain a 10% SDS-PAGE gel and rinse with deionized water. Remove the tape
and pull out the comb and put the gel in the gel box. Fill the upper buffer chamber with 1x running
buffer and allow gel to equilibrate for 10 minutes. Heat samples for 10 minutes at 70oC to break
disulfide bonds of proteins and then put the samples on ice. 25 µg of each protein sample gets
loaded into each well. This is equivalent to load 20 µl of each protein sample into the specific wells
and load a ladder in well 1. Each protein sample is run in two adjacent wells. Fill the lower buffer
chamber with running buffer and connect electrodes to the gel box. Run the gel at 200 volts for 35
minutes. After, shut the power off and disconnect electrodes. Separate the two cassette plates to
isolate the gel itself.
Obtain a piece of nitrocellulose membrane and presoak in transfer buffer. Place a presoaked
piece of filter paper on top of the gel and roll out any air bubbles. Place transfer membrane on the
gel and remove bubbles. Assemble the “gel sandwich” by placing blotting pads on the outsides and
inside between the filter papers of the two groups. Place the assembled unit into the buffer
chamber. Fill the blot module with transfer buffer and fill the outer chamber with deionized water.
Place lid on and run the gel at 30 volts for 80 minutes.
Part III: The membrane was placed in a 5% milk in TBST solution to block available sites
in the membrane’s pores. The membrane was rinsed and then placed in 1:1000 primary rabbit-antiBCL-2 antibody in 5% milk in TBST over night at 4oC. Wash the membrane in TBST 3 times for
10 minutes each by rocking the membrane for 10 minutes and then pouring out the TBST. After
being washed 3 times, add 10 mL of 1:2000 secondary goat anti-rabbit antibody bound with HRP,
horse radish peroxidase, in 5% milk in TBST and allow membranes to rock for 45 minutes at room
temperature. Wash membrane in TBST three more times and prepare 3 mL of developing solution.
Pipet onto the membrane and wait 5 minutes. Remove excess developing solution with a kimwipe
and examine membrane to visualize banding pattern.
Results: After examination in Part I of the experiment under the microscope, all the dishes of cell
culture samples had bands of cells that were visible at 400x magnification. The most “floaters” or
dead cells were in the 10% serum plus camptothecin. The other three of 2%, 5%, and 10% serum
all had cells adhered to the plating dish. Overall, the confluency of cells increased from 2% up to
10% FBS. Furthermore, the resulting gel after electrophoresis was examined in a dark chamber
where a camera caught the luminescence and yielded Figure 1. The cell cultures began growing and
developing in different environments where the variable was the serum they were kept in. The four
altered solutions were 2% FBS, 5% FBS, 10% FBS, and 10% FBS plus camptothecin.
Figure 1 illustrates the bands of BCL-2 proteins that are present in each cell culture sample.
Lanes 1 and 2 contain protein from the 2% serum; lanes 3 and 4 contain protein from the 5% serum;
lanes 5 and 6 contain protein from the 10% serum; and lanes 7 and 8 contain protein from the 10%
serum plus camptothecin (Table 1). The 10% serum has the bands of protein that have the greatest
intensity and are the thickest out of all four samples. Then, the 5% serum has the next thickest
bands of protein that are almost of the same intensity. The 2% serum and the 10% serum plus
camptothecin both have relatively thin bands of BCL-2 protein.
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Figure 1.
1 2 3
4 5
6 7 8
Figure 1. Western Blotting Results. Bands of BCL-2 protein are luminescing at different intensities
in each well. Every two wells correspond to a different sample of protein that was harvested from a
specific cell culture developed under a certain environment.
Table 1.
Lanes 1 and 2 2% serum
Lanes 3 and 4 5% serum
Lanes 5 and 6 10% serum
Lanes 7 and 8 10% serum + Camptothecin
Table 1. Legend for Figure 1. The four samples of proteins were run twice each and were harvested
from specific cells grown in a varying environment based on serum concentration.
Discussion: The purpose of this lab was to determine the levels of a protein called BCL-2 from the
cells cultured in different growth serum conditions. BCL-2 is a protein involved in the apoptotic
pathway which prevents cell death (Dressler et. al, 1993). This means that the presence of BCL-2
protein would yield an increased amount of cell survival. Furthermore, after the cells were grown
in 2% FBS, the confluency increased by only a small margin while the 5% FBS and 10% FBS cell
cultures had more significant increases in confluency with the 10% having the most cells.
However, the confluency of the cells for the 10% FBS plus camptothecin decreased overall since
the culture had some “floaters,” or dead cells, in the serum. These dead cells most likely died from
the addition of the camptothecin due to the chemical’s potential toxicity. Otherwise, the FBS
allowed for the growth, maintenance, and development of the cell cultures with the greatest
concentrations of FBS yielding an increased volume of cells and survival.
Likewise, cells cultured that did not have enough FBS or had the apoptotic inducer,
camptothecin, were predicted to have lower levels of the BCL-2 protein, while the cell cultures with
the greater FBS concentration and no camptothecin were predicted to have increased levels of BCL2. The results supported this hypothesis. After running the polyacrylamide gel, the 10% serum had
the thickest and most intense protein bands at the BCL-2 size (Figure 1). At the same time, the 2%
serum and the 10% serum plus camptothecin had the thinnest and faintest protein bands of the cell
culture samples (Figure 1). In essence, the data supports that the highest BCL-2 expression was in
the 10% FBS treatment whereas the lowest BCL-2 protein expressions were in the 2% and 10% +
Camptothecin treatments. These results make sense of the predicted hypothesis. The greater
concentration of FBS containing the greater amounts of necessary proteins, lipids, nucleotides, and
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other materials needed for cell growth, development, and function had the larger expression of the
BCL-2 protein. This higher protein expression was also connected with increased cell survival
which is supported by the observation that the 10% serum had the greatest confluency of cells.
The BCL-2 protein is a specific size no matter which cell culture it was synthesized in.
Therefore, the results show a line of bands all in a row which corresponds to the BCL-2 protein for
each cell sample (Figure 1). The row can be compared to the ladder in order to find the size of the
BCL-2 protein. Also, this protein is used in many scientific literature papers and is found to be
approximately 25 amino acids in size (Wang et. al, 2013).
Another protein to look at in these cells to see if the protein concentration differed would be
BAX. BAX is a protein that is like BCL-2 in that it is in the same family of proteins but differs in
that it is a pro-apoptotic protein (cite). Instead of monitor apoptosis and promoting cell survival like
BCL-2 does, BAX promotes the process of apoptosis in the presence of certain signals for cell
death. The BAX protein could produce results for an experiment of this kind that are opposite of
the BCL-2 observations. BAX could be measured using a Western Blot and bands of protein for
each culture would form at the specific size for BAX. We could then compare the cultures and
most likely notice that the 10% serum + Camptothecin would have the greatest BAX concentration
since camptothecin promotes cell death.
In addition, no distinct abnormalities occurred during the experiment. The results supported
the hypothesis and prediction. The only questionable abnormality would be that the confluency was
not very clear in determining the amount of cells that were still alive and functioning. There was
not a very distinct confluent difference between the cell culture samples. Furthermore, based on the
experiment, cells should be grown in a greater concentration of media such as the 10% FBS with no
other chemicals. This allows for substantial amounts of nutrients, proteins, and other necessary
developmental materials to be on hand for the cells in the culture. The more that the cells are
provided for, the greater potential for cell growth and ultimately survival.
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References
Dressler, F., J.A. Whalen, B.N. Reinhardt, & A.C. Steere. (1993). Western Blotting in the
Serodiagnosis of Lyme Disease. Journal of Infectious Diseases, 167(2), 392-400.
Irvine, J.J., G.J. Newlands, J.F. Huntley, & H.P. Miller. (1990). Interaction of Murine Intestinal
Mast Cell Proteinase with Inhibitors (Serpins) in Blood; Analysis by SDS-PAGE and
Western Blotting. Immunology, 69(1), 139-144.
Tsuji, T., R.M. Alborno, M. Ehara, T. Honda, & T. Miwatani. (1989). Detection of IgA Protease
from Haemophilus Influenzae by Immunoblotting. European Journal of Epidemiology,
5(2), 199-201.
Wang, Y., M. Li, W. Zang, Y. Ma, N. Wang, & G. Zhao. (2013). MiR-429 Up-Regulation
Induces Apoptosis and Suppresses Invasion by Targeting Bcl-2 and SP-1 in Esophageal
Carcinoma. Cellular Oncology, 36(5), 385-394.