Tasks
- Prepare Thermolysin Reactions
- 0.00063 g of Chymotrypsin dissolved in 1 mL of Tris/CaCl2 buffer (18.21 μM stock solution)
- 0.274(6) mL of stock solution + 4.725 mL of Tris/CaCl2 buffer --> 1 μM
- 0.027(4) mL of stock solution + 4.973 mL of Tris/CaCl2 buffer --> 100 nM
- 0.002(7) mL of stock solution + 4.997 mL of Tris/CaCl2 buffer --> 10 nM
- 0.027(4) mL of dilution (0.1821 μM) + 4.973 mL of Tris/NaCl CaCl2buffer --> 1 nM
- Fibers centrifuge (300 RPM for 10 min) + liquid was extracted.
- Different protease concentrations added + samples were left in the incubator shaker at 236 rpm (37C) for 2.5 hours.
- Collected 500 μL of sample at: 0, 5min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min, 90 min and 120 min.
- Prepare Lysozyme control
- 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 + 4.5 mL of water --> 2 - 5 mL samples of 5.56 μM lysozyme solution.
- solutions 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.
Note
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.
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