AU Biomaterials Design Lab:Protocols/PE Luminescence Spectrophotometer

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Contents

Description

Luminescence spectroscopy collects information on the way chemicals, that have been electronically excited, decay back to their ground state. Most often spectra include exciting at one specific wavelength and measuring the emission over a range of wavelengths. Luminescence covers several different kinds of excited state decays. We will most often be measuring fluorescence emission (emission from the singlet excited state). For biological studies, fluorescence is often used as a reporter of enzyme activity or presence or a reporter of protein structural properties. The fluorescent amino acid, tryptophan, has a different fluorescence emission in a folded state (when the tryptophan will likely be sequestered inside of the folded protein) than when the protein is unfolded (when the tryptophan will be more solvent exposed). The luminescence spectrophotometer is found in room 207.

Protocol

  1. If Necessary
    1. The instrument MUST be turned on before the computer. If the computer is already on, you will have to shut it down.
    2. Set up the peltier temperature control device.
    3. Open the program "FL WinLab"
  2. Measurement setup
    1. Click the spectrum icon on the top toolbar (a rainbow with a spectrum underneath)
    2. From the "Setup Parameters" tab, choose the type of measurement to preform
      1. Note: For most of what we do, this will be "Emission"
      2. Choose a start wavelength
        1. For tryptophan: 310 nm
      3. Choose an end wavelength
        1. For tryptophan: 500 nm
      4. Choose an excitation wavelength
        1. For tryptophan: 290 nm
      5. Set the excitation slit width (typical is 10 nm) It is important you note what you use for this value.
      6. Set the emission slit width (typical is 10 nm) It is important you note what you use for this value.
      7. Set the scan speed
        1. 100 nm/min is typical for our experiments. It is important you note what you use for this value.
      8. Enter a result filename. (The data are always automatically saved to: C:\flwinlab\data)
  3. Start your measurement
    1. Click on the green "Stop Light" icon.
  4. Saving your data (This part is convoluted)
    1. The data is automatically saved in a format that is proprietary to the instrumentation software. You need to save it in a format that is readable to you.
    2. In the FLWinLab window, expand the "Graph 1" window, you should see a graph displayed
    3. From the Graph 1 window menu, Choose File > Open from the menu
    4. Sort the names by date so your file is easier to find
    5. Select the file that you want to view and save and click "OK"
    6. Click on the name of your file (shown on the bottom of the graph window). This is especially important when there are multiple spectra shown in the same graph.
    7. From the Graph 1 window menu, choose File > Save As
    8. Change the file type to: ASCII
    9. Change the extension of the filename from ".sp" to ".txt"
  5. Collect more spectra as needed.
  6. Shut down the instrument
    1. Close all FLWinLab windows
    2. Turn off the instrument
    3. Turn off the computer


Notes

References

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