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:
- Used eppendorf tube no. 10 that weighed 1.02024g, and contained 0.00122g of trypsin.
- Added 1mL of phosphate buffer.
- Final concentration: 52.36µM
- Used 7 eppendorf tubes, each containing gold fibers.
- To each tube add:
- 0.9981mL of buffer
- 0.0019mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
- In 7 eppendorf tubes add to each:
- 0.9981mL of phosphate buffer
- 0.0019mL of trypsin solution
- Additional specifications:
- Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
- 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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