The following lab (Rubisco Western Blot) procedure was done over a course of three weeks.

During Week 1, gels were prepared, plant extracts were produced and the Bradford Assay was completed. First 2 identical gels were poured. This was done by assembling 2 sets of glass plates, inserting combs between the plates, and using a permanent marker to mark a line 1 cm below the teeth of the comb. The comb was then removed. Vinyl Gloves were put on for protection from acrylamide due to the toxicity of the liquid for of the substance. In a smaller flask, the components were mixed for the resolving gel in said order: 5.0 ml deionized water, 6.0 ml 30% acrylamide mix, 3.75 ml 1.5 M Tris (pH 8.8), 0.15 ml 10% SDS 0.15 ml, 10% APS 6 μl TEMED. Polymerization …show more content…
Using a p1000 micropipette, the gel was overlayed with a thin layer of DI water. Said steps were repeated for the second gel. The mixture was left at room temperature for 30 minutes allowing the mixture to completely polymerize. Any excess acrylamide in the mixing vessel was allowed to solidify before being thrown away. The top of the resolving gel was then rinsed with DI water and blot dried with a paper tower. Vinyl gloves were put back on for the next step. Using the same flask (cleaned) mentioned before, the following components of the stacking gel were mixed in said order: 3.4 ml deionized water, 0.83 ml 30% acrylamide mix, 0.63 ml 1.0 M Tris (pH 6.8), 0.05 ml 10% SDS, 0.05 ml 10% APS, 5 μl TEMED. The components were swirled and mixed using a plastic Pasteur pipet, the mixture was then added to the gel until it reached the top of the plate. The comb was then inserted and said steps were repeated for the second gel. The gel was the polymerized at room temperature for 45 minutes. Once gels were fully polymerized, they were then removed from the plastic clamp and each were …show more content…
The Pea plants were germinated and grown under 2 conditions: in light and complete darkness. 3 protein extracts were made, one from light-grown plants, a second from dark-grown plants, a third from dark grown plants exposed to 60 minutes of fluorescent light. During this incubation time, extracts of the other plant samples were made. The first extract was from the light-grown plants. 1 gram of light-grown plant leaf tissue was placed in a prechilled mortar, which sat on ice. 2 ml of cold extraction buffer was then added. The contents were then grinded with a pestle until a fluid slurry was present. The mortar was kept on ice through the procedure to ensure the extract remained cold and decreased the degradation of proteins by cell proteases. 2 microcentrifuge tubes as were labelled as “Light” and placed upon ice. All of the extract was then transferred into the tubes (making sure that the volume of extract in the tubes was about even). The tubes were then put on ice until they were centrifuged at 14,000 rpm for 5 minutes. A new microcentrifuge tube was labeled as “Light” and placed on ice. Using a micropipette, 400 μl of supernatant was carefully transferred to the new tube, avoiding the pellet. The same was done with the other tube of extract, such that a total of 800 μl of supernatant was pooled into one tube. The mortar and pestle were then rinsed with water and put back on ice. The same extraction