User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/11/10

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Objective

Measure protease Trypsin's kinetics with the Bradford Assay using 1 nM of protease (Trypsin) at different heating time intervals.

Procedure

The general protocol detailed in Dr. Hartings' notebook was used. The following specific steps were performed:

  • Protease Sample Prep.
  1. Used eppendorf tube no. (2) that weighed 1.02622g, and contained 0.00148g of trypsin.
  2. Added 1mL of Tris buffer.
  3. Final concentration: 63.52 µM
  4. We diluted this sample by pipetting 0.0157 mL and 0.9843 mL of Tris buffer to create a 1.0 µM Trypsin solution.
  5. We diluted this again sample by pipetting 0.050 mL and 0.950 mL of Tris buffer to create a 50 mM Trypsin solution.
  6. We pipetted 0.02 mL of the 50 nM solution with 0.98 mL of buffer to create to make a 1 nM Trypsin solution.
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers. (fibers made this way)
  2. To each tube add:
    1. 1 mL gold fibers
    2. 0.98 mL of buffer
    3. 0.02 mL of 1 nM trypsin solution (add this at the time of putting the tubes in the 37˚C hot water bath).
  • Blank Prep.
  1. In on eppendorf tube add:
    1. 0.98 mL of tris buffer
    2. 0.02 mL of 1 nM trypsin solution
  • Additional specifications:
  1. Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300 rpm.
  2. After heating the samples, they were all centrifuged for 1 min. at 13'200 rpm. This was done only for the samples, not the blanks.

Calculations:

   1st Dilution
   V1 = [(1µM)(1mL)]/63.52µM = 0.0157mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.0157mL = 0.9843mL
   2nd Dilution
   V1 = [(0.05µM)(1mL)]/1µM = 0.050mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.05mL = 0.95mL

Data

Graph 1nM Trypsin.Abs vs Wl.png

Graph 1nM Trypsin.Abs vs time I.png

Graph 1nM Trypsin.Abs vs time II.png

Graph 1nM Trypsin.Abs vs time III.png

Graph 1nM Trypsin.Abs vs time IIII.png

Results