User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/10/13

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Objective

Measure protease Trypsin's kinetics with the fluorescence assay using 1µM of trypsin at different heating 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. 5 that weighed 1.02922g, and contained 0.00181g of trypsin.
  2. Added 1mL of phosphate buffer.
  3. Final concentration: 77.68µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.987mL of buffer
    2. 0.013mL 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. 0.987mL of phosphate buffer
    2. 0.013mL 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 = [(1µM)(1mL)]/77.68µM = 0.01287mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.01287mL = 0.98713mL

Data


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