User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/25

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1 µM Trypsin In Situ Kinetics Protocol 90.7 µL of the stock Trypsin was diluted with 2.909 mL of Tris/NaCl for a final concentration of 1 µM. Fibers were spun yesterday and the liquid was extracted off the top. OceanOptics was run at 2 hours, scanning every 30 seconds. Results 100 nM Trypsin In Situ Kinetics Protocol 9.07 µL of the stock was diluted with 2.991 mL Tris/NaCl. Fibers were spun at 1500 RPM inadvertently. Same reaction conditions as above. Results Chymotrypsin Reaction Preparation 0.00060 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [trypsin] = 24.00 μM Bradford/Gel Analysis samples to be run: 1. 0.208 mL of stock solution in 4.792 mL of Tris/NaCl buffer [chymotrypsin]final = 1 μM 2. 0.020(8) mL of stock solution in 4.979 mL of Tris/NaCl buffer [chymotrypsin]final = 100 nM 3. 0.002(1) mL of stock solution in 4.998 mL of Tris/NaCl buffer [chymotrypsin]final = 10 nM 4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.24 μM Take 0.020(8) mL of dilution in 4.979 mL of Tris/NaCl buffer [chymotrypsin]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. A 500 μL aliquot was obtained every 5 minutes from the 1 μM and 100 nM samples. Timepoints including: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min. A 500 μL aliquot was obtained every 30 minutes from the 10 nM and 1 nM samples. Timpoints including: 0 min, 30 min, 60 min, 90 min, 120 min, 20 hrs, 21 hrs, 22 hrs, 23 hrs. The samples were shaken at 236 rpm and 37 C for the duration of the reaction time.