Week 5
We prepared ourselves for the p450 experiment by making 1 mol of KPO4, NADPH, G6PO4, G6PDH, and a 1:4 dilution of MgCl. Then we made our tubes in preparation for the experiment. We then set up charts of ‘time dependent’ and ‘protein dependent’ experiments. Today we proceeded to complete the experiment doing the time dependent after a lecture of the procedure. We only had time to run the timed experiment, so Dr. Clarke ran the protein experiment over night. We did a binding experiment with Dr. Harp. We talked about IC50 and how we were going to set up the experiment. Then we talked about how to set up the experiment with the tubes and the different concentrations of drug. Since we were not able to finish our own drugs, we used some that Dr. Harp already had for us. Then we proceeded to interpret the data from Dr. Harp’s experiment since we did not finish. Today Dr. Clarke told us that we were to do another experiment like the one from Monday and Tuesday, but on a smaller scale. We will be using different concentrations of DPP and of our individual drugs (Phenacetin, Dextromethorphan, ibuprofen, and Terpenadine). This experiment will tell us if DPP is an inhibiter of the drugs.
Week 4 with Dr. Harp
We preformed two tlc’s to determine our product but no such luck. So we had to use a column chromatography to determine if we had any product in out solution. Column Chromatography is just like tlc just that the produce travels downward due to gravity. Using tubes we collected samples of the solution to run tlc’s on the individual tubes. Today we flushed our column chromatography solution and rotervaped our completed DFB and prepared an NMR test solution. We learned about how we use an NMR to determine the products magnetic field and from that we can see what is in our solution. We went to wake forest to se a 300 mhz magnet that was used for the NMR. Then we went back to PTCRC to heir a lecture that Dr. Harp had to give. Today we helped clean the lab up and organize it a little. Then we proceeded to rotervap our flush and tube solution from the other day. Once completed, we prepared solutions for the rat dissection. Dr. Harp showed us how to kill the rat properly and the important parts of the dissection.
Week 3 with Dr. Clarke and Harp
We ran the Bradford protein assay again because the results were not reliable. Then we used the Genios machine to test the concentration of the enzymes. Last we compared and analyzed our data with one another. The nex day we helped Dr. Harp with children from a summer program making aspirin using salicylic acid and phosphoric acid as well. Then heated and filtered using filter paper. It was fun working with those kids. We helped Dr. Harp with the same kids,the next day, to separate the caffeine from tea. First we made the tea. Then we used dichloromethane and sodium hydroxide and magnesium sulfate to separate the caffeine and water from the tea. Then they scraped the caffeine and put it in to some vials and took them home. I've never work with kids that we that young and so many of them at the same time. With Doctor Clarke, we talked about Terfenadine, Dextromethorphan, S-ibuprofen, and Phenacetin. After making enough of each chemical for everyone, we picked who would do which and we poured some into vials. The amounts varied because we needed one mole of each and that is dependent on the molecular weight. Then we ran out of time so we stopped there to pick up at a later time.
