User:Beatriz Gimenez De C./Notebook/572/2015/02/18

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Tasks

  • Monitor through bradford the reaction of the AuNP fibers and 4 different concentrations of Trypsin.
    • 33.005 μM Trypsin stock solution was prepare. (0.00077 g of Trypsin dissolved in 1 mL of Tris/NaCl buffer)
    • Set 4 reactions and monitor every 30 min
      • 1 μM trypsine --> 0.151 mL of stock + 4.849 mL of Tris/NaCl buffer
      • 100 nM trypsine --> 0.015 mL of stock solution + 4.985 mL of Tris/NaCl buffer
      • 10 nM trypsine --> 0.002 mL of stock solution + 4.998 mL of Tris/NaCl buffer
      • 1 nM Trypsin --> 2 μL of stock solution + 4.9998 of Tris/NaCl buffer
    • Fibers centrifuge (300 RPM for 6 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 and 180 min
  • Monitor through UV-vis. (4 samples above diluted 10 times)
    • 10 nM trypsin --> 0.0907 mL of diluted stock in 2.909 mL of Tris/NaCl buffer
    • 1nM trypsin --> 0.00907 mL of diluted stock in 2.991 mL of Tris/NaCl buffer
    • all samples were introduced in Ocean Optics (scanned every 30 sec during 2 hours at 37°C and 1000 RPM)

Results

  • Trypsin kinetics through Ocean Optics

Image:10nM_ProtK_Kinetics_Chart.png

Image:10nM_ProtK_Kinetics_Abs.png




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