User:Beatriz Gimenez De C./Notebook/572/2015/02/10

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Tasks

  • Set and monitor Proteinase K Reaction
    • 0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM
    • Fibers centrifuge (300 RPM for 8 min) + liquid was extracted.
    • Different protease concentrations added + 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
  • Monitor through Bradford analysis samples
    • Samples run 150 min; extracting 300 μL samples every 30 minutes:
      • 0.116 mL of stock solution + 4.884 mL of Tris/NaCl buffer --> [proteinase K]final = 1 μM
      • 0.012 mL of stock solution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]final = 100 nM
      • 0.001(2) mL of stock solution in 4.999 mL of Tris/NaCl buffer --> [proteinase K]final = 10 nM
      • 10 μL of stock solution in 990 μL of Tris/NaCl buffer then take 0.012 mL of dilution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]final = 1 nM
  • Monitor with UV-Vis
    • Fibers centrifuged (300 RPM for 5 min) + liquid extracted.
    • Used Ocean Optics + set to scan every 30 sec for 2 hours (at 37°C + 1000 RPM).
    • 0.0075 mL of stock solution in 2.9925 mL of Tris/NaCl buffer --> [proteinase K]final = 100 nM
  • Bradford Calibration Curve
    • 100 μg/mL stock lysozyme + 0.023 mL of stock proteinase K (43.25 μM) --> [proteinase K] in lysozme stock = 100 nM
    • 6 samples were run in order to construct a calibration curve:
      • 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
      • 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
      • 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
      • 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
      • 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
      • 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

Results

  • Absorbance vs Time for the Proteinase K In situ reaction.

  • Kinetics at 517.7 nm.

  • Absorbance vs Time for the Proteinase K In situ reaction.

  • Bradford Results

Image:Bradford_Calibration_Curve_Feb10-NEW.png


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