User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/11/10

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Objective Measure protease Trypsin's kinetics with the Bradford Assay using 1 nM of protease (Trypsin) at different heating time intervals. Procedure The general protocol detailed in Dr. Hartings' notebook was used. The following specific steps were performed: Protease Sample Prep. 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. Sample Prep. 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). Blank Prep. 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. Calculations: 1st Dilution V1 = [(1µM)(1mL)]/63.52µM = 0.0157mL, amount of trypsin solution needed Volume of buffer: 1mL - 0.0157mL = 0.9843mL 2nd Dilution V1 = [(0.05µM)(1mL)]/1µM = 0.050mL, amount of trypsin solution needed Volume of buffer: 1mL - 0.05mL = 0.95mL Data Results