James Madison University
BIO 480 Webposter
Methods
Plant Sample Collection
Three Chrysanthemum x morifolium plants were purchased. Each of the plants chosen was at a different stage in growth of its flowers. The first was budding, the second was beginning to bloom and the third was fully bloomed while approaching senescence. The night prior to laboratory, leaves were trimmed from each plant (samples 1,2,3), and stems were trimmed from plants two and three (samples 4,5). All samples were kept frozen, separately, until the next day. Then they were taken into the laboratory and frozen there until use.
Analysis of RBCL Protein Expression
Protein Isolation and Analysis
Protein was isolated from all five samples by cutting and grinding plant tissue, with buffer added. This slurry was then centrifuged to remove bulky tissues. Supernatants from all samples were analyzed to find concentration using the BioRad’s DC protein assay. We set up a series of standards for the creation of standard curve, which were assayed in the same way as the five samples. All were tested for concentration at 750nm. Protein samples were stored for future use in Western blotting.
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In step 8C, the following volumes were used: 12.5 mL Reagent A and 250 µL Reagent S to create reagent SA. |
Polyacrylamide Gel Electrophoresis (PAGE) and Western Blot
A quantity of 30 µg of each protein sample was run on two identical SDS-PAGE gels. The first SDS-PAGE was stained using Comassie Blue dye and photographed using BioRad imager. A rainbow molecular weight marker was used for comparison to determine the size of protein in our samples. The second SDS-PAGE was used for electroblotting. Proteins from the gel were transferred onto an Immobilon-PDVF transfer membrane using electrophoresis. The membrane was stored until Western Blotting analysis.
Antibody Detection and Western Blot Analysis
The membrane prepared by electroblotting protein samples from our SDS-PAGE gel was probed using a sequence of two antibodies, primary chicken anti-Rubisco antibody followed by secondary goat anti-chicken antibody. This prepared the membrane for a luminescent reaction using luminol/enhancer to detect Rubisco large subunit protein.
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Alternative Procedure was used, therefore in steps 2 and 5, the antibody was diluted in pure TTBS. |
Analysis of RBCL DNA
Genomic DNA Extraction and Analysis
Genomic DNA (including both chloroplast and chromosomal DNA) was extracted and isolated from all five samples of chrysanthemum. Spectrophotometric readings at 280 nm and 260 nm were taken to analyze samples for both purity and concentration. Once sample concentrations were determined, the volume of each sample was calculated to generate 50 ng of DNA per sample. These were the volumes used in real time PCR.
Real-Time Polymerase Chain Reaction (PCR)
Real-Time PCR was performed on all five DNA samples to amplify the RBCL region of chloroplast DNA which encodes the Rubisco protein. The amounts of amplified DNA will be compared between samples.
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RBCL2F forward primers and RBCL-fonfana reverse primers were used. |
Agarose Gel Electrophoresis of PCR product DNA
DNA fragments generated by PCR were separated on a 2% (w/v) agarose gel. They were compared with a 100bp DNA ladder to estimate the size of the fragments separated on the gel and to determine if the RBCL region of DNA, which encodes the Rubisco protein, was amplified.