User:Asya L. Tucker/Notebook/Asya 571/2015/09/09

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Objectives

  1. To determine the molar absorptivity of lysozyme using UV-Vis. This data is useful as we move on to protein concentration with the Bradford assay in the future.
  2. To determine how protein concentration affects fluorescence intensity.
  3. Prepare a protease sample.

Procedure

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 Fluorescence.
    3. Correct the spectra for solvent and baseline
  1. Fluorescence 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. Mass of 1 eppendorf tube: 1.03215 g
    2. (Add roughly 1 mg of Trypsin) Mass of Trypsin: 0.00122 g
    3. Label the tube with the name of the protease and what the concentration will be after you add 1 mL of water.
      1. mass of trypsin/23300(MW of Trypsin)/.001 L =__ M
    4. Place in your freezer box

Data

RAMG2 Lysozyme Fluorescence Graphsept9.png

Lysozyme Molar Absorbtivity Graph.RAM.png


Results

Lysozyme Molar Absorbtivity Coefficient: 37700 M-1 cm-1