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.

10nM ProtK Kinetics AbsvsTime Chart.png

10nM ProtK Kinetics Chart.png


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