User:M Jaffe/Notebook/Maxis Protease Lab 471/2015/09/30

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Objectives

  1. To make standardized curves for determining peptide concentration using fluorescence analysis.
  2. To run blanks for UV/Vis experiments we did yesterday.

Procedure

Fluorescence EXPERIMENT

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

To perform a protease digest of lysozyme

  1. To make a stock lysozyme solution we weighed out 5.14 mg of lysozyme and placed it into a 5 mL volumetric flask and pipetted to the line with phosphate buffer.
  2. We retrieved one of our pre-measured protease (trypsin) and added 1 mL phosphate buffer to create a 53.22 µM protease (trypsin) stock.
  3. We combined 0.0188 mL of the 53.22 µM protease stock with 0.9812 mL stock lysozyme solution to create a 1 µM protease reaction sample solution.
  4. We combined 0.0188 mL of protease stock with .9812 mL of phosphate buffer to create a blank sample (no lysozyme).
  5. We placed both tubes (reaction sample and blank sample) to a 37C water bath for 1hr
  6. To make standards for analysis
    1. Standard A: 150uL of reaction sample we
    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. To make blanks for analysis we
    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
  8. To create samples for measurement we
    1. For each of the standards and blanks described above mix the following
        • 20uL of sample
        • 140uL of Assay Buffer
        • 40uL of Assay Reagent
  1. We took measurements at
        • Excitation at 390 nm
        • Emission from 400 to 650 nm

UV/Vis EXPERIMENT

  1. We followed the procedure here except we did not use any gold fibers.

Data