User:Asya L. Tucker/Notebook/Asya 571/2015/10/14

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Objective

Measure protease Trypsin's kinetics with the Bradford Assay using 100 nM of protease 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. 6 that weighed 1.02625 g, and contained 0.00128 g of trypsin.
  2. Added 1mL of Tris buffer.
  3. Final concentration: 54.93 µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers. (fibers made this way)
  2. To each tube add:
    1. 0.99818 mL of buffer
    2. 0.00182 mL 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. 0.99818 mL of tris buffer
    2. 0.00182 mL of 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 13'200rpm. This was done only for the samples, not the blanks.

Calculations:

   V1 = [(0.1µM)(1mL)]/54.93µM = 0.00182mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.00182mL = 0.99818mL

Data

Results


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