User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/18

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10 nM Protease K Kinetics Run

Objective

Determine the kinetics of Protease K on AuNP fibers at 10 nM enzyme concentration

Protocol

69 μL of the 0.4325 μM stock was diluted with 2.931 mL of Tris/NaCl buffer. This was added onto the fibers in the cuvette. 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.

10nM ProtK Kinetics AbsvsTime Chart.png

10nM ProtK Kinetics Chart.png

Determination of Trypsin Cleavage of AuNP Fibers

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