June 19, 2014
Data Analysis:
- Axes under layout
- Double click to change values on axes
- To save graphs as pictures, right click and do move to new sheet, copy it and paste it in paint, and save as a .png
- To import text filed from uv vis, go do data > from text > import > next > comma (to separate into two columns) > finish
- To correct based on water reference, import all necessary data, make a new column for Abs., hit =, select the absorbance minus the water reference, drag down as far as you want to extend the calculation
- To get chart to open wetware, copy and paste chart into paint, save it as a .png, and put it with the rest of the data in Dr. Hartings's drop box >look under toolbox in wetware, and slect upload file, hit upload file, and hyperlink it to the notebook using [ [ link ] ] without spaces
- When copying and pasting link, only use the portion of the link after the standard openwetware piece that is on every link to the site
- to edit the size of the graph, put a vertical bar like [ [ link | size ] ] without spaces
UV Vis Initial Setup Instructions
- When initially set up, click connect in the bottom of the screen to allow it to warm up
- Then do edit > method > make a new folder for today in Dr. H's dropbox
- Put the water reference into the spec, and hit the green button on the keyboard (F9)
- When next sample is put in, do method > filename, and change the name
- Once it has run, save it as a .spc file within the program
- After each run, the cuvette was thoroughly rinsed with distilled water, and completely dried. Excess water was removed with a glass pipette
- When done, hit disconnect and turn off
Results of Dialysis:
- Fluid from dialysis tubing was removed from the first tube by pouring it, but after a slight spill occurred, a syringe was used to remove the fluid from the remaining dialysis tube
- Dialyzed apoprotein was allowed to sit on ice while awaiting spectroscopy
- Spectrophotometry revealed for the pH 2.5 sample that, although a large amount of protein was lost, protein was still present in the solution
- Similar results were observed for the pH 3.0 sample
- Slightly more protein was present in the less acidic solution however
- Since so much protein is being lost during dialysis, we will try to use a desalting column to slowly raise the pH
Extraction Using 2 mL 1M HCl:
- Same procedures previously described were used, and similar results were obtained
- Differences in procedure: acid was added dropwise using a 10μL pipette until the desired pH (roughly 2.8) was achieved. pH strips were used to estimate
Results from Spectrophotometer:
- It appeared as if heme was still present in the apoprotein, judging from a peak around 485
- Another extraction needed to be done
Lyophilizer:
- KCl + NaOH + Myoglobin
- Sample was places in a Falcon tube, and played in a bed of dry ice. Sample was completely frozen in around 30 minutes
- The cap on the tube was replaced with a special one with small holes in the cap, and moved to the lyophilizer overnight
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