User:Rebeca Rodriguez/Notebook/Chem 471/2015/10/14

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Fluorescence Analysis of Protease Degradation

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

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

All samples will be incubated in eppendorf tubes, which will be placed in the 37 °C water bath on the shaker. Samples should all contain the same concentration of materials.

You should make samples for measurements at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr.

  1. Protease Prep
    1. Place the 1mL of phosphate buffer in the pre-measured proteinase K sample and record the concentration (3.70·10-5 M).
  2. Sample Prep
    1. To a fiber sample tube (that has been centrifuged for 10 minutes at 300 RPM and had the supernatant removed) add the appropriate amounts of buffer and Proteinase K (2.7 μL of Proteinase K, 997.3 μL Buffer)
      1. The total volume should be 1mL
      2. The final protease concentration should be 100nM
      3. Vortex the sample to disperse the fibers
  3. Blank Prep (you will have one blank to match each sample)
    1. To a clean eppendorf tube add the appropriate amounts of buffer and protease (2.7 μL of Proteinase K, 997.3 μL Buffer)
      1. The total volume should be 1mL
      2. The final protease concentration should be 100nM
  4. Incubate Sample and Blanks
    1. Place the tubes (for both the Blanks and Samples) in the shaker with the 37°C water bath
  5. Measurement
    1. Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time
    2. For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube
      1. Blank measurement
        1. From the blank measurement, take 20uL of blank and place in a 600uL eppendorf tube
        2. Add 140uL of Assay Buffer
        3. Add 40uL of Assay Reagent
        4. Take Measurement
      2. Sample Measurement
        1. From the sample, take 20uL of sample and place in a 600uL eppendorf tube
        2. Add 140uL of Assay Buffer
        3. Add 40uL of Assay Reagent
        4. Take Measurement
    3. Excitation at 390 nm
    4. Emission from 400 to 650 nm

Data

The integration values were then converted to concentration values using the calibration curve created on 9/29.


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