User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/24

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March 24th, 2015

Proteinase K Reaction Preparation

0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM


Bradford/Gel Analysis samples:

1. 0.116 mL of stock solution in 4.884 mL of Tris/CaCl2 buffer

  • [proteinase K]final = 1 μM

2. 0.012 mL of stock solution in 4.988 mL of Tris/CaCl2 buffer

  • [proteinase K]final = 100 nM

3. 0.001(2) mL of stock solution in 4.999 mL of Tris/CaCl2 buffer

  • [proteinase K]final = 10 nM

4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> take 0.012 mL of dilution in 4.988 mL of Tris/CaCl2 buffer

  • [proteinase K]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 at varying time points (depending on concentration) beginning with time point 0. The samples were kept in the incubator shaker (37C) at 236 rpm.

Time points collected:

  • 1 μM: 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 min
  • 100 nM: 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 min
  • 10 nM: 0, 30, 40, 50, 60, 70, 80, 90 min
  • 1 nM: 0, 30, 40, 50, 60, 70, 80, 90 min

NOTE: While all 5 mL of the 1 μM and 100 nM samples were depleted, the 10 and 1 nM reactions were left in the incubator/shaker overnight in order to extract samples the next day.

1 nM Chymotrypsin Kinetics

10.4 μL of dilute Chymotrypsin stock was diluted by 2.989 mL of buffer. Fibers and OceanOptics were prepared with the same protocol as previous runs.

Results

1nM Chymotrypsin AbsvsTime Mar24 Chart.png

1nM Chymotrypsin Kinetics Mar24 Chart.png

There were still fiber aggregates in solution when the reaction finished. No visible colorimetric change occurred.