User:Asya L. Tucker/Notebook/Asya 571/2015/10/20

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Objective

To measure protease kinetics with the Fluorescence Assay using 1uM of protease.

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. 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 7 eppendorf tubes add to each:
    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

Results


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