Objective
Measure protease Trypsin kinetics with Bradford Assay using 0.1µM of trypsin.
Procedure
In order to do the Bradford Assay of Protease Degradation, we followed the general protocol detailed in Dr. Hartings' lab notebook, but with these changes:
- The wanted concentration of trypsin in each eppendorf tube had to now be 100nM (0.1µM) rather than 1µM.
- Protease Sample Prep.
- Used eppendorf tube no. 6 that weighed 1.02625g, and contained 0.00128g of trypsin.
- Added 1mL of Tris buffer.
- Final concentration: 54.93µM
- Used 7 eppendorf tubes, each containing gold fibers.
- To each tube add:
- 0.9982mL of buffer
- 0.0018mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
- In 7 clean 1mL eppendorf tubes add:
- 0.9982mL of tris buffer
- 0.0018mL 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.
- The Bradford dilution was 1:4; with 1mL of Bradford and 3mL of buffer.
Calculations:
V1 = [(0.1µM)(1mL)]/54.93µM = 0.0018mL, amount of trypsin solution needed
Volume of buffer: 1mL - 0.0018mL = 0.9982mL
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