User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/10/07

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The goal of the day was to make a calibration curve for the Fluorescence Analysis of Protease Degradation.


The instructions for the day can be found here

  1. Prepping the Lysozyme
    1. Made a stock solution of Lysozyme by adding 1.24 mg to 1 mL of 50 mM pH 8 phosphate buffer
    2. Lysozyme concentration: (1.24 mg) / (1 mL) = 1.24 mg/mL Lysozyme
  2. Preparing Protinase K solution
    1. Protinase K tube was mixed with 1 mL of 50 mM pH 8 phosphate buffer
    2. Protinase K conentration: (0.00085g)*(1mol/28,900g)*(1/0.001L)= 0.0000294118 M Protinase 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 heated on a heat block at 37°C for an hour
  4. Making 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
    9. Make blanks for analysis
    10. Blank A: 150uL of blank sample
    11. Blank B: 75uL of Blank A and 75uL of water
    12. Blank C: 75uL of Blank B and 75uL of water
    13. Blank D: 75uL of Blank C and 75uL of water
    14. Blank E: 75uL of Blank D and 75uL of water
    15. Blank F: 75uL of Blank E and 75uL of water
    16. Blank G: 75uL of Blank F and 75uL of water
    17. Blank H: 75uL of Blank G and 75uL of water
  5. Measuring samples
    1. To each standard or blank was added 20uL of sample/blank, 140uL of Assay Buffer, and 40uL of Assay Reagent
    2. Measurements were taken with Excitation at 390 nm and Emission from 400 to 650 nm


Lysozyme concentration in A

  • (1.24 mg/mL)(0.966 mL) = (M2)(0.2 mL)
  • M2 = A = 0.119784 mg/mL

The actual concentrations of each sample is as follows

  • A: 0.119784 mg/mL
  • B: 0.059892 mg/mL
  • C: 0.029946 mg/mL
  • D: 0.014973 mg/mL
  • E: 0.0074865 mg/mL
  • F: 0.00374325 mg/mL
  • G: 0.001871625 mg/mL
  • H: 0.000935813 mg/mL

The image below shows the intensity for the fluorescence of samples A though H and blanks A through H from 400 to 650 nm. 2015 10 7 Fluorescence of Proteinase K.png

To create the calibration curve the samples and blanks had the area of their intensity integrated from 420 nm to 650 nm. The equation (Wavelength2 - Wavelength1)/2*0.5 was used and the sums of each interval totaled. The samples were then corrected for by subtracting area of the blank from the total area of the sample. The graph below shows the corrected integrated area from 420 to 650 nm vs concentration of the samples in mg/mL. A linear trend was created, with the line starting at the origin of the graph. The resulting linear trend was y = 688379x or intensity = 688378 * concentration. The R2 value was 0.9702. 2015 10 7 Lysozyme Fluorescence Calibration Curve of Proteinase K.png