R u b i s c o
METHODS
Our research began by isolating protein from four different ages of Arabidopsis thaliana; four weeks, six weeks, eight weeks, and ten weeks old. Here is the protocol we followed in performing this protein extraction. Instead of using 2 mL QB Buffer, 200 mL was used. Reagent SA (a copper containing reagent was made by mixing 10 mL Reagent A with 20 mL Reagent S. This was performed so that we could uses the protein at a later time for our Western Blot and our SDS-PAGE.
The next step was to isolate DNA from the same ages of tissue for use in the qPCR procedure. Here is the protocol we followed for the DNA extraction. Please note that step 13 was skipped.
Using the DNA received from the extraction performed, qPCR was used to analyze the relative amount of rubisco large-subunit coding DNA in vivo. Here is the qPCR protocol that we followed. Please note that the qPCR was stopped before it completed all cycles.
The products from the qPCR were then ran on an agarose gel to check for purity. Here is the agarose gel protocol. In part B, we did not use a restriction digest, but the samples from our qPCR reactions.
The protein isolated previously was used to run a western blot and an SDS-PAGE gel that were ran concurrently. Here is the protocol for the western blot and SDS-PAGE. Note that the only deviation form the protocol was that instead of using transfer buffer to transfer the protein to the membrane we used TTBS.
After the western blot has been performed, antibodies were used to detect the presence of rubisco large-subunit. Here is the protocol for antibody detection. We used 3 mL of our primary antibody and 1 mL of our secondary antibody.