User:Lymperopoulos Loukas/Notebook/471 LAB/2015/09/30

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Objective

  • To make standardized curves for determining peptide concentration using fluorescence analysis.

Description

  • Dr. Hartings left a procedure for us to use today in his lab 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 (~ 1mg/mL) using phosphate buffer
    2. Make your protease stock by adding 1mL to your pre-measured protease
    3. Make a sample that has a final volume of 1mL and protease concentration of 1uM
      1. Add the appropriate volume of protease sample to a clean eppendorf tube
      2. Add a volume of lysozyme solution so that the final volume is 1mL
    4. Make a blank sample (no lysozyme)
      1. Add the appropriate volume of protease sample to a clean eppendorf tube
      2. Add a volume of buffer solution so that the final volume is 1mL
    5. Add both tubes to the 37C 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

Data

Image:Lysozyme flourescences calobration curve.PNG

Notes

This area is for any observations or conclusions that you would like to note.


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