Rubisco
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Discussion
First, If there is a chance to pick which leaves are used in the experiment, DON'T PICK THE MAGNOLIA LEAF. It will take a very long time to grind in a mortar. Only pick the magnolia leaf if there is lots of time to kill.
Our hypothesis, which was that thicker leaves would contain more Rubisco, was not conclusive because of the unavailability to test all of the plant samples in the same way. For example the only plant samples used in the western blot and DNA electrophoresis was the magnolia sample. We were able to locate Rubisco in the magnolia sample, yet no other plant sample was tested because of the poor PCR results that we got with the other samples. We can not draw conclusion to the hypotheses, even though over all protein concentration was calculated. However, the other steps leading to our data is discussed as followed.
First a DC protein assay of the ground plant samples was ran at 750nm, concentrations of the proteins for each plant were figured out and recorded. It was found that for our samples red maple had a concentration of 37.99mg/ml for red maple, 13.365mg/ml for magnolia, 7.55 for tulip tree and 6.582 for the sycamore. With this calculation the amount of protein that would need to loaded into the western blot that would be done in a lab later in the experiment. Next DNA was isolated and ran in a spectrophotometer at 260nm and 280nm to find the 260/280 concentrations of DNA in our samples so that calculations could be made to determine the amount of DNA needed in the PCR experiment and how much needed to load into an agarose gel electrophoresis, they were as followed: red maple 520ug/ul, magnolia 322.5ug/ul, tulip 254.5 ug/ul, and sycamore 112.5ug/ul. This part of the experiment went well, other than the grinding of the magnolia sample which took for ever to do.
Even though the calculations and the loading of the PCR were done with minimal error, the PCR results may be faulty due to experimental error and also, some of the primers used might not have worked for the particular plants we were using. As seen in the peak in figure 1, magnolia was the only one which gave decent results. Therefore, magnolia was the only sample of DNA that was run during the agarose gel electrophoresis as seen in figure 3, which shows that the 200bp gene for rubisco is present in lane five.
The western blot was a success, we were able to isolate the protein rubisco’s large and small subunits which were located at the 300 and 500 marks of the molecular ladder in the picture of the western blot, which was taken from the membrane in which the electroblotting technique was performed as seen in figure5. The gel stained in comassie blue stain showed the presence of Rubisco also, as seen in figure 4.
The antibody detection was successful in marking the rubisco in the magnolia sample as seen in figure 5. We used chicken and rabbit antibody to find the rubisco in the magnolia sample.
We were unable to test our hypothesis to the extent that we wish we could, the experiment was a success for at least one of our samples, (magnolia) in which we were able to detect both DNA for the rubisco gene and the protein itself. Even though it is disheartening not to be able to proclaim the hypothesis null or void, the experience of doing the techniques performed in lab, and the fact that the techniques worked for one of our samples out weighs not knowing if thicker leaves contain more rubisco or not. Its is left up to other experimenters to find out now.