User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/09/09

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Objective

Determine the molar absorptivity of lysozyme. We will use this information as we move on to quantifying protein concentration with the Bradford assay.

Also determine how protein concentration affects fluorescence intensity.

Description

Group 2 will be working with the Ocean Optics spectrometer today.

Groups 1,3,4, and 5 will be doing fluorescence, luminescence, prepping protease samples, and starting to process fiber samples for mass analysis.

Groups 1, 3, 4, and 5 will all make their lysozyme samples at the beginning of class. Groups 1 and 3 will use the spectrometers first until all of they have data recorded for all of their samples. Groups 4 and 5 will work on prepping protease samples and processing fiber samples first until groups 1 and 3 are finished. After this point, groups 4 and 5 will work on spectroscopy while groups 1 and 3 prep protease samples.

NOTE: You need to use volumetric flasks (10 mL) when making all of your samples for spectroscopy today.

Protocols

  1. Lysozyme samples for spectral analysis
    1. Lysozyme has a molecular weight of 14307 g/mol
    2. Make 10 mL of roughly 50 uM lysozyme in water (it doesn't have to be perfect. you just HAVE to accurately know what it is)
    3. Save this stock concentration (in a 15 mL falcon tube) and rinse out your volumetric flask well with deionized water
    4. Make 5 lysozyme samples for analyzing with UV Vis and fluorescence spectroscopies. The concentrations should be between 0 and 15 uM.
      1. Be sure to rinse your flask well between making each sample.
  2. UV Vis data
    1. Measure a water blank
    2. Measure the spectrum of each of your samples (except for your 50 uM sample)
    3. Correct the spectra for solvent and baseline
  3. 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.
  4. 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
  5. 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.

Data

  • Add data and results here...

Notes

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


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