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METHODS
Collection of Bundle Sheath Cells: The plants used in this
experiment were gathered from the JMU Biology Department's
greenhouse located beside Burruss Hall with the help of Dr. Brubaker.
The bundle sheath cells were shaved from the underside of the C3
leaves using a razor blade, and from the inner part of the fleshy
CAM plants. Two grams were collected of each sample and used in the
first part of the experiment.
  
Protein Isolation:
The DC Protein Assay (BioRad) based on the Lowery Assay was used to
determine the protein concentration of our samples. This in turn was
used to determine the amount of protein to be used in the Western
Blot procedure. The protein isolation procedure was used to create a
standard curve of the protein concentration of the samples versus
their absorbance at 750nm. The curve was then used to convert the
absorbances into protein concentrations.
DNA Extraction: This protocol
allowed us to break open the cells and isolate genomic DNA. This DNA
was precipitated and its absorbance was measured to determine the
concentration of the DNA. This DNA was used in the next step of the
experiment, Real Time PCR. *Only three of our samples showed a
significant concenration of DNA, therefore we decided to eliminate
Hosta fortunei from our experiment.
Real Time PCR: In
using Real Time PCR, we were able to verify the presence of the gene
for Rubisco. The PCR product was detected using the sybr green dye
which fits between the bases of double stranded DNA. Primer set C
was used.
Agarose Gel Electrophoresis of DNA:
The agarose gel electrophoresis allowed us to separate the DNA
fragments produced by the previous procedure, Real Time PCR.
Western Blot: There were two parts
to this procedure. The first part used SDS- PAGE(Polyacrylamide Gel
Electrophoresis) to separate the proteins based on their size alone.
The negatively charged SDS in the procedure coats the individual
proteins which negates their charge. The proteins are also denatured
at 100 degrees Celsius with a reducing agent present. A marker is
used to determine the size of the proteins. The second part of the
Western Blot calls for the transfer of the proteins on the gel onto
a membrane. This membrane will be used in the subsequent detection
of the Rubisco protein using antibodies.
Antibody Detection: This procedure
uses primary and secondary antibodies to detect the Rubisco protein.
The primary antibody was chicken anti- Rubisco. This antibody was
made against a conserved region of the large subunit of the Rubisco
protein. The secondary antibody was made in a goat and binds to all
chicken antibodies. The secondary antibody was chemically coupled to
alkaline phosphatase which breaks down a colorless substrate into a
purple substance. This color change allows us to have some idea of
how much protein is located in a particular plant.
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