User:M Jaffe/Notebook/Maxis Protease Lab 471/2015/10/20

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Objective

Measure protease Trypsin's kinetics with the fluorescence assay using 100nM of trypsin at different heating intervals.

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. 10 that weighed 1.02024g, and contained 0.00122g of trypsin.
  2. Added 1mL of phosphate buffer.
  3. Final concentration: 52.36µM
  • 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

RAMfluorescence100nm.png