User:Andrew W Long/Notebook/Protein Modification/2014/06/19

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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

Apo mb postdialysis 6-19.png

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