User:Sydney Marshall/Notebook/Protease Project/2015/10/14

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Objective

  • To analyze the digenstion of Proteinase K at 100 nM using fluorescence assay

Description

Full protocol can be found in Dr. Harting's notebook here.

  • Prepping Fiber Samples
    • 7 fiber samples were obtained and spun at 300 RPM for 10 min
    • The supernatant was removed
    • Proteinase K tube 11 was mixed with 1 mL of 50 mM Tris/10 mM CaCl2 pH 8 buffer
    • Proteinase K concentration: (0.00107g)*(1mol/28,900g)*(1/0.001L)= 0.00003702 M Proteinase K
    • Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (37.02 μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.0027 mL
    • Amount of Buffer solution need to get to 1mL: (1mL total)-(0.0027 mL Proteinase K solution) = 0.9973 mL buffer
  • Incubating Samples
    • 2.7 μL Proteinase K and 997.3 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

Image:2015 10 14 Fluorescence of Raw Data.png
This graph shows the samples and blanks from A-H for fluorescence assay from 400nm to 650nm.

Image:2015 10 14 Fluorescence Concentrations.png
This graph shows the concentration in 100 nm Proteinase K over time with the fluorescence assay.


Description

Full protocol can be found in Dr. Harting's notebook here.

  • Prepping Fiber Samples
    • 7 fiber samples were obtained and spun at 300 RPM for 10 min
    • The supernatant was removed
    • Proteinase K tube 11 was mixed with 1 mL of 50 mM Tris/10 mM CaCl2 pH 8 buffer
    • Proteinase K concentration: (0.00107g)*(1mol/28,900g)*(1/0.001L)= 0.00003702 M Proteinase K
    • Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (37.02 μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.0027 mL
    • Amount of Buffer solution need to get to 1mL: (1mL total)-(0.0027 mL Proteinase K solution) = 0.9973 mL buffer
  • Incubating Samples
    • 2.7 μL Proteinase K and 997.3 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


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