User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/29

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Fluorescence Analysis of Protease Degradation

The general procedure for today was taken from Dr. Hartings's Notebook.

We are using the Pierce quantitative fluorometric peptide assay. (product link here)

  1. Perform a protease digest of lysozyme
    1. Make a stock lysozyme solution (1.04 mg/mL) using phosphate buffer
    2. Make your protease stock by adding 1mL to your pre-measured protease (0.85 mg Proteinase K, 2.9381×10-5 M)
    3. Make a sample that has a final volume of 1mL and protease concentration of 1uM
      1. Add the appropriate volume of protease sample (0.034 mL Proteinase K) to a clean eppendorf tube
      2. Add a volume of lysozyme solution (0.96 mL Lysozyme) so that the final volume is 1mL
    4. Make a blank sample (no lysozyme)
      1. Add the appropriate volume of protease sample (0.034 mL Proteinase K) to a clean eppendorf tube
      2. Add a volume of buffer solution (0.96 mL buffer) so that the final volume is 1mL
    5. Add both tubes to the 37°C water bath for 1hr
    6. 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
    7. 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
  2. Make Samples for Measurement
    1. For each of the standards and blanks described above mix the following
      1. 20uL of sample
      2. 140uL of Assay Buffer
      3. 40uL of Assay Reagent
  3. Take Measurement
    1. Excitation at 390 nm
    2. Emission from 400 to 650 nm

Data

The Fluorescence calibration curve was created by subtracting the blank spectra from each of the respective standard spectra. The spectra were then integrated using the midpoint method and half wavelength intervals.




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