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Biology 241
Dr. Busch
12/06/2013
Introduction
Previous Research. Presently in the United States, cancer is the second leading cause of
death; therefore, researchers are pursuing knowledge in the field of chemotherapy and
chemoresistance (Leading Causes of Death 2013). Cisplatin, also known as CDDP, is a common
drug used to treat cancer. Researchers have discovered that the protein c-Jun is the regulator of
CDDP effectiveness. If there is a lot of active c-Jun, CDDP is less effective; however, if c-Jun is
inactive, CDDP is more effective. Researchers have conducted numerous experiments that
demonstrate the differing effects of cisplatin on cancer cells found in the brain, bone marrow, and
nervous system (Xia 2013). Cisplatin is not the only chemotherapy drug. Oxaliplatin is a
platinum-based drug that affects c-Jun levels colorectal cancer cells. Whether oxaliplatin has a
positive or negative effect on c-Jun levels in colorectal cancer cells, however, is unknown.
Proposed Objective. In order for the hypothesis to be tested, both gel electrophoresis and
western blotting will be utilized. Gel electrophoresis uses both an electric current and agarose gel
to separate segments of protein for this particular experiment (Gel Electrophoresis 2008).
Western blotting uses gel electrophoresis to separate proteins which are then transferred onto a
membrane and stained with specific antibodies (US National Library of Medicine National
Institutes of Health 2012). Both gel electrophoresis and western blotting aid in the determination
Proposed Research
Methodology. HCT116 adenocarcinoma cancer cells line the tissue of the human colon,
and these colorectal cancer cells will be used in the experiment (US National Library of
Medicine National Institutes of Health 1993). The experimental system will consist of HCT116
cells at a density of 4 10 per 60-mm plate. The cells will be grown on an agarose plate for 16
5
hours and treated with 50 g/ml oxaliplatin (Xia 2013). Organizing the HCT116 cells at a density
of 4 10 per 60-mm plate and allowing the cells to be treated for 16 hours ensures sufficient cell
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growth. Three plates will be treated with the solution of oxaliplatin. Three additional plates will
be used as a control group, treated without the solution of oxaliplatin. After 16 hours has passed,
a sample from each plate will be transferred into its own centrifuge tube and centrifuged for 10
minutes (US National Library of Medicine National Institutes of Health 2012). Centrifugation is
the spinning process used to separate materials suspended in a liquid medium. The pellet at the
bottom of the centrifuged tube contains c-Jun protein and will then undergo gel electrophoresis.
Gel electrophoresis will separate segments of protein based on size and charge. The protein
solutions will be placed in agarose wells, put into a box filled with buffer solution, and then
hooked up to an electric current. The buffer contains ions which allow the electricity to actually
pass through the apparatus, and the current causes the proteins to be pulled down the gel towards
the positive electric terminal (Gel Electrophoresis 2008). Upon completion of gel
electrophoresis, a western blot will be used. Western blotting enables the identification of c-Jun
from a complex mixture of proteins extracted from the pellet. The technique used to accomplish
western blotting began with gel electrophoresis which separated the proteins by size. Then the
obtained results will be transferred onto a membrane and incubated for 30 minutes on ice to
prevent the proteins from denaturing. Then specific antibodies are labeled to target the protein c-
Jun. The antibodies used for this experiment are Ser63, which is a primary antibody, and Ser73,
which is the secondary antibody. The primary antibody is added to the serum on the membrane
and incubated overnight in a cold environment. Then the membrane is washed and the secondary
antibody is injected into the serum and incubated for an hour. This incubation process ensures
that the targeted protein, c-Jun, is properly tagged. An enhanced chemiluminescence (ECL) mix
is prepared, which causes certain proteins to luminesce in dark conditions. The ECL is also
added to the serum and incubated for 1-2 minutes. Finally, the membrane is observed in a dark
Expected Results. Following observation of the western blot in a dark environment, some
bands on the membrane will glow and other bands will not glow. The bands containing
oxaliplatin glow less or not at all because oxaliplatin decreases c-Jun levels in colorectal cancer
cells (Xia 2013). Therefore, the hypothesis that oxaliplatin decreases c-Jun levels in colorectal
Gel Electrophoresis [Internet]. HudsonAlpha Institute for Biotechnology; [2008 Dec 12, cited
http://www.hudsonalpha.org/education/kits/hnpcc/electrophoresis
Leading Causes of Death [Internet]. 2013 Jan 11. Hyattsville(MD): Centers for Disease Control
and Prevention; [2013 Jan 11, cited 2013 Dec 5]. Available from:
http://www.cdc.gov/nchs/fastats/lcod.htm
US National Library of Medicine National Institutes of Health [Internet]. 2. 1993. PubMed [cited
US National Library of Medicine National Institutes of Health [Internet]. 9. 2012. PubMed [cited
Journal of Biological Chemistry (288) [Internet]. [2013 Jul 5, cited 2013 Dec 5]. Available
from: www.jbc.org/content/288/27/19321.full