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

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Procedure

  • We took the absorbtivity of lysozyme with UV VIS and Fluorescence More detailed instructions can be found here

Protocols

  1. Lysozyme samples for spectral analysis
    1. Lysozyme has a molecular weight of 14307 g/mol
    2. We prepared a 51.2uM lysozyme stock soluton by weighing out 7.32 mg of lysozyme and transfered it to a 10 mL volumetric flask where we pipetted to the mark with deionized water. We transfered our stock solution to a falcon tube.
    3. We made 5 lysozyme samples for analyzing with UV Vis and fluorescence spectroscopies between a working range on 0 and 15 uM.
      1. (volume of stock or previous sample)(Conc of used sample

0/(10 mL deionized water)=new sample conc.

      1. (2.93 mL)(51.2 uM)/10 mL= 15 uM
      2. (5 mL)(15 uM)/10 mL= 7.5uM
      3. (7 mL)(7.5 uM)/10 mL=5.25 uM
      4. (5 mL)(5.25 uM)/10 mL= 2.625 uM
      5. (3 mL)(2.625uM)/10 mL= 0.7875 uM
      6. UV Vis data
    1. We measured a water blank
    2. We ran our 5 samples within the working range, using the UV Vis and Flurescence.
    3. Correct the spectra for solvent and baseline
  1. Flurescence data
    1. Measure a water blank
    2. Measure the spectrum of each of your samples (except for your 50 uM sample)
    3. Using Excel, calculate the area underneath the curve (i.e. integrate) for each of your spectra.
  2. Prepping protease samples
    1. Record the mass of 1 eppendorf tube (This is important. We will use this data for something later on) and then TARE the balance
    2. Add roughly 1 mg of your protease to the tube and record the mass
    3. Label the tube with the name of the protease and what the concentration will be after you add 1 mL of water.
    4. Place in your freezer box
  3. Processing the fiber samples
    1. Centrifuge the fiber samples
    2. Carefully draw off the liquid supernatant (without disturbing the fibers). Its OK if you leave some water on the fiber.
    3. Leave the tubes open so that the water can evaporate.
    4. We will analyze these samples on a later date.

Method

  • Insert content here...

Data

RAMG2 Lysozyme Fluorescence Graphsept9.png

Lysozyme Molar Absorbtivity Graph.RAM.png

Matt Hartings Change the A vs Conc graph so that your data shows up as points, not as a curve. What is the line fit that you made? What is your calculated molar absorptivity in M-1 cm-1