Measure protease Trypsin's kinetics with the Bradford Assay using 10 nM of protease (Trypsin) at different heating time intervals.
The general protocol detailed in Dr. Hartings' notebook was used. The following specific steps were performed:
- Used eppendorf tube no. 2 that weighed 1.02622 g, and contained 0.00148 g of trypsin.
- Added 1mL of Tris buffer.
- Final concentration: 63.52 µM
- We diluted this sample by pipetting 0.0157 mL and 0.9843 mL of Tris buffer to create a 1.0 µM Trypsin solution.
- We pipetted 0.01 mL of the 1.0 µM solution with 0.99 mL of buffer to create to make a 0.01 µM Trypsin solution.
- Used 7 eppendorf tubes, each containing gold fibers. (fibers made this way)
- To each tube add:
- 1 mL gold fibers
- 0.99 mL of buffer
- 0.01 mL of 0.01 µM trypsin solution (add this at the time of putting the tubes in the 37˚C hot water bath).
- In on eppendorf tube add:
- 0.99 mL of tris buffer
- 0.01 mL of 0.01 µM trypsin solution
- Additional specifications:
- Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300 rpm.
- 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.
V1 = [(0.01µM)(1mL)]/63.52µM = 0.000157mL, amount of trypsin solution needed
Volume of buffer: 1mL - 0.000157mL = 0.9843mL
V1 = [(1.0µM)(1mL)]/63.52µM = 0.0157mL, amount of trypsin solution needed
Volume of buffer: 1mL - 0.01mL = 0.99mL