User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/18

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February 18th, 2015 Trypsin Reaction Preparation 0.00077 g of Trypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [trypsin] = 33.05 μM Bradford/Gel Analysis samples to be run for three/four hours; extracting 500 μL samples every 30 minutes: 1. 0.151 mL of stock solution in 4.849 mL of Tris/NaCl buffer [trypsin]final = 1 μM 2. 0.015(1) mL of stock solution in 4.985 mL of Tris/NaCl buffer [trypsin]final = 100 nM 3. 0.001(5) mL of stock solution in 4.998 mL of Tris/NaCl buffer [trypsin]final = 10 nM 4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.3305 μM Take 0.015(1) mL of dilution in 4.985 mL of Tris/NaCl buffer [trypsin]final = 1 nM Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube and a 500 μL aliquot from each concentration was obtained every 30 minutes beginning with time point 0. The samples were left in the incubator shaker (37C) for 3.0 hours. Time points collected: 0, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min In-situ UV-Vis samples: Today's work: 69 μL of the 0.4325 μM Proteinase K stock was diluted with 2.931 mL of Tris/NaCl buffer for a final [Proteinase K] of 10 nM. This was added onto the fibers in the cuvette, which were spun down at 300 RPM for five minutes, liquid extracted, and resuspended. The reaction was run at 37°C for 6 hours, scanning every 5 minutes. Results The reaction did have a discernible colorimetric change. There were still fibers present in solution after 6 hours. A longer reaction may see this go to completion. ALL FROM 1:10 DILUTION - See sample 4 above! The following are calculations for the future; these experiments have not been executed! 1. 0.0907 mL of diluted stock in 2.909 mL of Tris/NaCl buffer [trypsin]final = 10 nM 2. 0.00907 mL of diluted stock in 2.991 mL of Tris/NaCl buffer [trypsin]final = 1 nM