Measure protease Trypsin's kinetics with the Bradford Assay using 1 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 1.02622g, and contained 0.00148g 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 diluted this again sample by pipetting 0.050 mL and 0.950 mL of Tris buffer to create a 50 mM Trypsin solution.
- 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.
- Used 7 eppendorf tubes, each containing gold fibers. (fibers made this way)
- To each tube add:
- 1 mL gold fibers
- 0.98 mL of buffer
- 0.02 mL of 1 nM trypsin solution (add this at the time of putting the tubes in the 37˚C hot water bath).
- In on eppendorf tube add:
- 0.98 mL of tris buffer
- 0.02 mL of 1 nM 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 = [(1µM)(1mL)]/63.52µM = 0.0157mL, amount of trypsin solution needed
Volume of buffer: 1mL - 0.0157mL = 0.9843mL
V1 = [(0.05µM)(1mL)]/1µM = 0.050mL, amount of trypsin solution needed
Volume of buffer: 1mL - 0.05mL = 0.95mL