Methods
The samples used in this experiment were from the genera Chrysanthemum and Dianthus taken from common house plants. This genera of plant was used because they seem to be among one of the more common types of plants sold for a home garden. The parts of the plant that were analyzed were leaves, stems, and petals.
Chrysanthemum Dianthus
Protein
Approximately 1g of cut plant tissue was grinded into a mortar with about 2ml of cold extraction QB buffer (100mM potassium phosphate buffer, 1mM EDTA, 1% Triton-X-100, and 10% glycerol with some protease inhibitors, pH 7.8) and a pinch of sand. Supernatant was stored after samples were centrifuged at top speed for 15 minutes. This was repeated for all samples (leaves, petals, stems of Chrysanthemum and Dianthus).
DC protein assay for protein concentration determination:
DC protein assays were run using the diluted protein to determine the concentration of protein standard. Spectrometry was used to read absorbance at 750nm.
Western blot analysis was used for further protein analysis with the preparation of two PAGE gels. The first was to run a native PAGE gel under high pH conditions to separate proteins by their surface charge and their size. This was stained with comasie blue. The other gel was used for SDS PAGE, and then those proteins were transferred to a nitrocellulose membrane using electroblotting techniques for further use in analysis using antibodies. A rainbow molecular weight marker was used.
Antibody Detection and Western Blot Analysis:
The protein in the membrane made from the previous week was detected using two antibodies. The primary antibody was the large subunit of Rubisco protein made from a chicken, by Agrisera. The secondary antibody used was a goat antibody which binds to the primary antibody. It also binds to alkaline phosphatase to create a colorimetric or luminescent reaction for detection. Dilutions were 1:20,000 for both the primary and secondary antibodies. The alternative procedure for light reaction was used for this experiment.
DNA
Extraction, Purification and Concentration Determination:
DNA was extracted from 1-3grams of plant tissue with the use of chloroform/isoamyl alcohol (octanol) (24:1) to be used later analysis.
Absorbances of samples were taken at 260nm and 280nm on a spectrophotometer for DNA purity and concentration determination. Purity was found by A260/A280 and concentrations were found by Beer’s Law.
PCR was performed on 100ug of DNA plant tissues to determine the amount of RBCL gene for each sample. Forward and reverse primers used for amplification were rbcl2F (RbclReiseberg) and RBCL-Savolainen.
Agarose gel electrophoresis was used to separate DNA fragments made during PCR for the determination of the presence of Rubisco DNA. 2% agarose gels were made in 1X TAE. Tracking dyes that were used in the samples were xylene cyanol FF 4kB and bromophenol blue 300bp. The molecular weight marker used was 100bp DNA ladder.