User:Sydney Marshall/Notebook/Protease Project/2015/10/07

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Objective

  • Make a calibration curve for Fluorescence Analysis of Protease Degradation.

Description

The product link for the assay can be found here)

  1. Preparing lysozyme
    1. Make a stock lysozyme solution (1.24mg/1mL) using 50 mM pH 8 phosphate buffer
  2. Preparing proteinase K
    1. Proteinase K tube was mixed with 1 mL of 50 mM pH 8 phosphate buffer
    2. Proteinase K conentration: (0.00085g)*(1mol/28,900g)*(1/0.001L)= 0.0000294118 M Proteinase K
    3. Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (29.4118 μM)*(V1) = (1 μM)*(1 mL) => V1 = 33.99 μL Proteinase K
    4. Amount of Lysozyme stock needed to reach 1 mL: (1mL total)-( 33.99 μL Protinase K solution) = 966.01 μL Lysozyme
  3. Preparing samples
    1. 966 μL Lysozyme and 34 μL Proteinase K were mixed together for the sample tubes and 966 μL phosphate buffer and 34 μL Proteinase K were mixed together for blanks
    2. All samples were incubated at 37°C for an hour
  4. Make standards for analysis
    1. Standard A: 150uL of reaction sample
    2. Standard B: 75uL of Standard A and 75uL of water
    3. Standard C: 75uL of Standard B and 75uL of water
    4. Standard D: 75uL of Standard C and 75uL of water
    5. Standard E: 75uL of Standard D and 75uL of water
    6. Standard F: 75uL of Standard E and 75uL of water
    7. Standard G: 75uL of Standard F and 75uL of water
    8. Standard H: 75uL of Standard G and 75uL of water
  5. Make blanks for analysis
    1. Blank A: 150uL of blank sample
    2. Blank B: 75uL of Blank A and 75uL of water
    3. Blank C: 75uL of Blank B and 75uL of water
    4. Blank D: 75uL of Blank C and 75uL of water
    5. Blank E: 75uL of Blank D and 75uL of water
    6. Blank F: 75uL of Blank E and 75uL of water
    7. Blank G: 75uL of Blank F and 75uL of water
    8. Blank H: 75uL of Blank G and 75uL of water
  6. Make Samples for Measurement
    1. 20uL of sample of each, 140uL of Assay Buffer, 40uL of Assay Reagent were placed in the quartz cuvette and their measurements were taken.
  7. Take Measurement
    1. Excitation at 390 nm
    2. Emission from 400 to 650 nm

Results

2015 10 7 Fluorescence of Proteinase K.png
These are the curves for the samples and blanks for fluorescence assay or proteinase K from 400nm to 650nm.

2015 10 7 Lysozyme Fluorescence Calibration Curve of Proteinase K.png
This is the calibration curve for the Fluorescence Assay of Proteinase K with lysozyme.