Methods
Protein Isolation : (Complete Protocol)
Protein was isolated from each plant sample. Two grams of green leaf and two grams of pigmented flower were collected from the purple, orange, and white Chrysanthemums.
For the DC Protein Assay, a total of twenty five tubes were set up- those used for the series of standards (7) as well as three tubes for each sample (18). The absorbance of the samples was compared to the standards to determine the concentration in the samples. Absorbance readings were taken at 750nm. It was important for the absorbance readings of each sample to be above 0.125 mg/ml and below 1.25 mg/ml so that these values would fall on the standard curve. In order to assure this, the proteins samples were made up of 1:2, 1:20, and 1: 200 dilutions in QB buffer. The absorbance readings of the 1:200 dilutions from the petal and leaf samples fell within the standards range, so those values were used to calculate the protein concentration in each undiluted sample.
The concentrations of each sample found during the DC Protein assay ultimately determined how much protein was used for Western Blotting later in the experiment.
DNA extraction: (Complete Protocol)
DNA was isolated from each plant sample from both the leaf and flower tissues. In this step frozen plant tissue was ground up in CTAB reagent. (100mM Tris pH= 8.0, 1.4M NaCl, 25 mM EDTA, 2% CTAB, 0.2% beta-mercaptoethanol) The DNA isolated in this step of the procedure was used for the PCR reaction.
Real Time PCR: (Complete Protocol)
Before conducting the PCR reaction, the concentration and purities of DNA samples from the leaf and flower tissue had to be found. To determine DNA purity, the absorbance of each sample was taken at 260nm and 280nm. An A280/A260 value of 1.8-2.0 indicates a pure DNA sample. If this ratio is lower, that indicates there is some protein contamination in the sample. One can use the A260 value to find the concentration of DNA in each sample. Concentration= A260 x 1/bε where 1/bε= 50 µg/mL
Real Time PCR was used to quantitate the amount of genomic RUBISCO in each sample. 25ng and 50ng of each DNA sample was loaded into the PCR reaction mix along with one No DNA control.
Following the PCR reaction, a 2% agarose gel was run to separate the DNA fragments generated by the PCR reaction and to confirm the presence of the RUBISCO gene. The original gel was loaded with DNA generated from the 25ng PCR sample. Unfortunately, the buffer used while running this gel was a 50X TAE buffer, when the protocol called for a 1X. A new gel with DNA generated from the 50ng PCR samples with the correct buffer. (Agarose Gel Protocol)
The gel results of each sample were compared with a 1kb ladder and no DNA control in adjacent lanes. The gel was stained with ethidium bromide to visualize the DNA bands.
Western Blot: (Complete Protocol)
Two gels were run using the isolated protein from plant tissue collected earlier in the experiment. 30µg of each protein sample, a positive control, and molecular weight marker were loaded into a 10% acrylamide gel and underwent SDS page electrophoresis. The gel was later stained with Comassie blue. SDS page rids the proteins of their negative charge and separates the proteins on the gel based on size alone. Comassie blue sticks to aromatic amino acids within proteins, helping to stain and make them visible on the gel. This gel was used to determine the presence of the RUBISCO large subunit in the samples.
The second 10% acrylamide gel was made. 30µg of each protein sample, a positive control, and a molecular weight marker were loaded into the lanes of the gel and underwent SDS page electrophoresis. However, instead of staining the gel with Comassie blue, the gel was electroblotted.
After electroblotting, the gel was exposed to antibodies in order to detect the RUBISCO large subunit.
Antibody Detection and Western Blot Analysis : (Complete Protocol)
Two different antibodies, a primary and a secondary, were used to detect the RUBISCO protein. The primary, anti-RUBISCO chicken antibody was expected to bind to conserved regions of the large subunit of RUBISCO. The secondary antibody, produced in goats, binds to all chicken antibodies. The second antibody was chemically linked to the enzyme horseradish peroxidase which causes a colorimetric reaction to occur once bound to the primary antibody. The colorimetric reaction enabled us to detect the RUBISCO product.
In step two and five of the protocol, a primary antibody dilution of 1:20,000 in 20mL of blocking buffer (TTBS) was used. An alternate procedure was followed and dry milk was not added to the diluted solution.