User:Asya L. Tucker/Notebook/Asya 571/2015/10/07

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Objective

Measure protease Trypsin kinetics with Bradford Assay using 1µM of trypsin.

Procedure

In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' protocol, and did the following specific steps:

  • Protease Sample Prep.
  1. Used eppendorf tube no. 11 that weighed 1.02097g, and contained 0.00154g.
  2. Added 1mL of Tris buffer.
  3. Final concentration: 66.09µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.985mL of buffer
    2. 0.015mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
  • Blank Prep.
  1. In 7 clean 1mL eppendorf tubes add:
    1. 0.985mL of tris buffer
    2. 0.015mL 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.
  3. The Bradford dilution was 1:4; with 1mL of Bradford and 3mL of buffer.

Calculations:

   V1 = [(1µM)(1mL)]/66.09µM = 0.01513mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.01513mL = 0.98487mL

Data

Graph 1uM Trypsin.Abs vs Wl.png

Graph 1uM Trypsin.Abs vs Time I.png

Graph 1uM Trypsin.Abs vs Time II.png

Results