User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/10

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February 10th, 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 to be run for three/four hours; extracting 300 μL samples every 30 minutes: 1. 0.116 mL of stock solution in 4.884 mL of Tris/NaCl buffer [proteinase K]final = 1 μM 2. 0.012 mL of stock solution in 4.988 mL of Tris/NaCl buffer [proteinase K]final = 100 nM 3. 0.001(2) mL of stock solution in 4.999 mL of Tris/NaCl 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/NaCl 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 300 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 2.5 hours. Time points collected: 0, 30 min, 60 min, 90 min, 120 min, 150 min In-situ UV-Vis samples: 1. 0.0075 mL of stock solution in 2.9925 mL of Tris/NaCl buffer [proteinase K]final = 100 nM Fibers were spun down at 300 RPM for 8 minutes, and liquid extracted; the fibers were resuspended and added to the cuvette. The Ocean Optics was set to scan every 30 seconds for 2 hours, and maintained at 37°C and stirred at 1000 RPM. In-situ Results Absorbance vs Time for the Proteinase K In situ reaction. Kinetics at 517.7 nm. Notice the plateau between 90 minutes and 120 minutes. This is seen in the absorbance vs time chart as well. Absorbance vs Time for the Proteinase K In situ reaction. Bradford Calibration Curve To the 100 μg/mL stock lysozyme solution prepared last week, 0.023 mL of stock proteinase K (43.25 μM) was added in order to cleave the protein into fragments. [proteinase K] in lysozme stock = 100 nM 5 samples were run in order to construct a calibration curve: 1. 0.025 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.775 mL Tris/NaCl buffer [lysozyme] = 2.5 μg/mL 2. 0.05 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.750 mL Tris/NaCl buffer [lysozyme] = 5.0 μg/mL 3. 0.10 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.700 mL Tris/NaCl buffer [lysozyme] = 10.0 μg/mL 4. 0.125 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.775 mL Tris/NaCl buffer [lysozyme] = 12.5 μg/mL 5. 0.15 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.650 mL Tris/NaCl buffer [lysozyme] = 15.0 μg/mL 6. 0.20 mL of stock lysozyme || 0.2 mL 1:4 Bradford:Tris/NaCl || 0.600 mL Tris/NaCl buffer [lysozyme] = 20.0 μg/mL NOTE: Tomorrow, 250 μL samples of each proteinase K reaction at 30 min time points will be run with 200 μL of 1:4 Bradford:Tris/NaCl and 1550 μL of Tris/NaCl buffer. (because full volume cuvettes)