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

1uM Trypsin AbsvsTime Feb25.png

1uM Trypsin Kinetics Chart Feb25.png

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

100nM Tryspin AbsvsTime Feb25 Chart.png

100nM Tryspin Kinetics Feb25 Chart.png

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.