Methods
Rubisco was isolated from the leaf, stem, and root of the elephant ear plant In order to determine how much protein was present in our samples, we first needed to grind the leaves into a slurry using a mortar and pestle with 2mL of protein isolation buffer QB (100mM potassium phosphate buffer pH = 7.8, 1mM EDTA, 1% Triton-X-100, and 10% glycerol). The stem was difficult to grind, so more QB buffer was used. Then, a DC protein assay was run to determine the concentration of protein in our samples. The absorbance of each sample at 750nm was compared to a standard curve in order to determine the concentration. The concentrations of the protein samples were found using the equation from the graph.
Next, 50mL of the ground up leaf, stem, and root samples was placed in a 50oC water bath with CTAB buffer in order to lyse the membrane of the plant cells. Once the DNA and the protein were exposed, chloroform/isoamyl alcohol was added to each sample. This binds to the proteins and polysaccharides. The solution separated into a lower phase (chloroform/octanol, proteins, and carbohydrates) and an upper aqueous phase (DNA). The sample was precipitated using isopropanol and then centrifuged. The upper layer containing the DNA was transferred to a 25mL tube. Beer’s law was then used to determine the concentration by measuring the absorbance at 260nm and 280nm.
Primer set A (rbcl2f and RBCL-fonfana) and Primer set C (rbcl2f and RBCL-Savolainen) were selected in order to isolate and amplify the segment of DNA which contains the gene for Rubisco. Each DNA sample (50 ng) was added to a master mix containing primers A and C, both of which are universal primers. A 500bp and a 200bp fragment were expected once run on a gel. Duplicates of each sample were made for the Reverse Transcription PCR. The RT-PCR was carried out using a computer program and the data was used to verify the presence of the gene for Rubisco in each sample.
Agarose Gel of Real Time- 09/26/05
After the desired cDNA was amplified with PCR, Agarose Gel Electrophoresis was used to separate the fragments. Twenty-five mls of 2% (w/v) solution of agarose in TAE buffer was made and the solution was allowed to set. The tracking dye, bromphenol blue in a 50% glycerol solution, was added to each sample. Then, each sample and a molecular weight marker were loaded. After the gel finished running, it was stained using Ethidium Bromide. A picture was then taken using the Bio-Rad Chemilumenescent camera. It was expected that primer A would show bands at approximately 500bp and Primer C should show bands at approximately 200bp.
Two pre-made SDS polyacrylamide gels were used in order to separate proteins. Gels were shared between two groups and both gels were made identical to each other. The negatively charged SDS molecules coat the proteins, causing them to migrate towards the positive end of the gel. The larger molecules remain at near the top of the gel by the wells, and the smaller molecules migrate farther towards the bottom of the gel. The first gel was stained with Comassi Blue over night and photographed. The second gel was used in the Western Blot. The next day, the membrane from the Western blot was washed in a buffer solution and then stored in a blocking solution (25ml TTBS, 2.5g non-fat dry milk) for one week.
The second gel from the western blot was transferred to a membrane. During the antibody detection, the rubisco band could be specifically located on the membrane. By exposing the membrane to chicken antibodies (antibodies that bind with the rubisco) and then exposing the membrane to rabbit antibodies (which are anti-antibodies to the chicken antibodies), the resulting signal allows the visualization of the rubisco protein on the membrane. The membrane was washed using an alternative protocol which used 15ml of TTBS and 3µl of Chicken anti RBCL. The second wash was done using 15ml of TTBS and 1µl of rabbit anti-chicken with horse radish peroxidase. The blot was then incubated for five minutes in 6 ml of luminol/enhancer and 6 ml of peroxide buffer. This was so that any remaining antibody signal would luminesce off of the membrane when a picture was taken. The membrane was covered in saran wrap and a picture was taken with the BioRAD Chemilumenescent camera.
Introduction Results Discussion
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