User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/04

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March 4th, 2015

Thermolysin Reaction Preparation

0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/CaCl2 buffer ---> stock [thermolysin] = 18.21 μM

Bradford/Gel Analysis samples to be run:

1. 0.274(6) mL of stock solution in 4.725 mL of Tris/CaCl2 buffer

  • [thermolysin]final = 1 μM

2. 0.027(4) mL of stock solution in 4.973 mL of Tris/CaCl2 buffer

  • [thermolysin]final = 100 nM

3. 0.002(7) mL of stock solution in 4.997 mL of Tris/CaCl2 buffer

  • [thermolysin]final = 10 nM

4. 10 μL of stock solution in 990 μL of Tris/CaCl2 buffer --> [trypsin] = 0.1821 μM

    • Take 0.027(4) mL of dilution in 4.973 mL of Tris/NaCl CaCl2buffer
  • [thermolysin]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 for all protease concentrations every 5 minutes until 30 minutes, timepoints including: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min. A 500 μL aliquot was obtained every 30 minutes after the first 30 min until 2 hours of total reaction, timepoints including: 60 min, 90 min, 120 min. The samples were shaken at 236 rpm and 37 C for the duration of the reaction time.

Lysozyme control digestion

55.6 μM lysozyme stock solution was prepared from 0.079 g of lysozyme in 100 mL of water.

0.5 mL of this stock solution was combined with 4.5 mL of water in order to produce 2 - 5 mL samples of 5.56 μM lysozyme solution. These were digested with thermolysin protease with total solution protease concentrations of 1 and 100 nM. See above procedure for thermolysin stock volumes resulting in given concentrations.

Samples were mistakenly pulled at the same timepoints as the thermolysin/AuNP fiber reactions, but only the 120 min timepoint will be run on the gels.

1 μM Chymotrypsin Kinetics

Protocol

Switching to Chymotrypsin kinetics. Same protocol for OceanOptics, scan every 30 seconds for 2 hours at 37°C, spinning at 1000 RPM.

For the solution prep, 104 μL of concentrated Chymotrypsin stock (24.00 μM) was diluted by 2.896 mL of Tris-10mM CaCl2 buffer.

Results



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