User:Asya L. Tucker/Notebook/Asya 571/2015/11/10

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Objective

Measure protease Trypsin's kinetics with the Bradford Assay using 1 nM of protease (Trypsin) 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. (2) that weighed 1.02622g, and contained 0.00148g of trypsin.
  2. Added 1mL of Tris buffer.
  3. Final concentration: 63.52 µM
  4. We diluted this sample by pipetting 0.0157 mL and 0.9843 mL of Tris buffer to create a 1.0 µM Trypsin solution.
  5. We diluted this again sample by pipetting 0.050 mL and 0.950 mL of Tris buffer to create a 50 mM Trypsin solution.
  6. We pipetted 0.02 mL of the 50 nM solution with 0.98 mL of buffer to create to make a 1 nM Trypsin solution.
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers. (fibers made this way)
  2. To each tube add:
    1. 1 mL gold fibers
    2. 0.98 mL of buffer
    3. 0.02 mL of 1 nM trypsin solution (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. 0.98 mL of tris buffer
    2. 0.02 mL of 1 nM 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 13'200 rpm. This was done only for the samples, not the blanks.

Calculations:

   1st Dilution
   V1 = [(1µM)(1mL)]/63.52µM = 0.0157mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.0157mL = 0.9843mL
   2nd Dilution
   V1 = [(0.05µM)(1mL)]/1µM = 0.050mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.05mL = 0.95mL

Data

Results


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