User:Asya L. Tucker/Notebook/Asya 571/2015/11/04

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Objective Measure protease Trypsin's kinetics with the fluorescence assay using 1nM of trypsin at different heating intervals. Create a calibration curve using fluorescence for trypsin. Procedure The general protocol detailed in Dr. Hartings' lab notebook was used. The following specific steps were performed: Protease Sample Prep. Used eppendorf tube no. 6 that weighed (1.01922 )g, and contained (0.00128)g of trypsin. Added 1mL of phosphate buffer. Final concentration: (54.93 µM We diluted the 54.93 uM )Trypsin sample by pipetted 0.0182 mL to 0.982 ml of phosphate buffer to make 1 uM solution of Trypsin. We pipetted 0.01 mL to make 0.01 uM solution of Trypsin. Sample Prep. 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). Blank Prep. In on eppendorf tube add: 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