User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/11/04

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Objective

  1. Measure protease Trypsin's kinetics with the fluorescence assay using 1nM of trypsin at different heating intervals.
  2. Create a calibration curve using fluorescence for trypsin.

Procedure

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

  • Protease Sample Prep.
  1. Used eppendorf tube no. 6 that weighed (1.01922 )g, and contained (0.00128)g of trypsin.
  2. Added 1mL of phosphate buffer.
  3. Final concentration: (54.93 µM
  4. We diluted the 54.93 uM )Trypsin sample by pipetted 0.0182 mL to 0.982 ml of phosphate buffer to make 1 uM solution of Trypsin.
  5. We pipetted 0.01 mL to make 0.01 uM solution of Trypsin.
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.9981mL of buffer
    2. 0.0019mL of trypsin (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. 0.9981mL of phosphate buffer
    2. 0.0019mL of trypsin solution
  • Additional specifications:
  1. Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
  2. After heating the samples, they were all centrifuged for 1 min. at 13'200rpm. This was done only for the samples, not the blanks.

Calculations:

   V1 = [(0.1µM)(1mL)]/52.36µM = 0.0019mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.0019mL = 0.9981mL

Data

RAM fluorescence1nmnov4.png