User:Sydney Marshall/Notebook/Protease Project/2015/10/28

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Objective

To analyze the digestion of Proteinase K at 1 μM using fluorescence assay

Description

  • Prepping Fiber Samples
    • 7 fiber samples were obtained and spun at 300 RPM for 10 min
    • The supernatant was removed
    • Proteinase K tube 1B was mixed with 1 mL of 50 mM Tris/10 mM CaCl2 pH 8 buffer
    • Proteinase K concentration: (0.00166g)*(1mol/28,900g)*(1/0.001L)= 0.0000574 M Proteinase K
    • Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (57.4 μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.017 mL
    • Amount of Buffer solution need to get to 1mL: (1mL total)-(0.017 mL Proteinase K solution) = 0.983 mL buffer
  • Incubating Samples
    • 17 μL Proteinase K and 983 μL buffer were mixed in the 7 fiber tubes as well as 7 blank eppendorf tubes
  • Eppendorf tubes were incubated on a shaker in a 37°C water bath for 10 min, 15 min, 30 min, 45 min, 1 hrs, 1.5 hrs, and 2 hrs for one of the fiber tubes and one of the blanks.
  • Running Samples
    • Each sample was centrifuged at 12000 rpm for 1 min to collect the remaining fibers
    • For each sample/blank, 20uL was taken and placed in a 600uL eppendorf tube along with 140uL of Assay Buffer and 40uL of Assay Reagent.
  • Taking Measurement
    • Excitation at 390 nm
    • Emission from 400 to 650 nm

Results

2015 10 28 Fluorescence of Raw Data.png
This graph shows the fluorescence curves for the samples and their respective blanks. 2015 10 28 Fluorescence Concentrations.png
This graph shows the concentration of 1uM proteinase K n mg/ml over time using the calibration curve for the proteinase K with lysozyme.