User:Anneliese Faustino/Notebook/671 Nanoparticles/2016/09/07

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Protocol for Fluorescence Apparatus

  1. Starting the Peltier controller
    1. Make sure that the ribbon cord connects the peltier controller and the cuvette holder
    2. Fill the chiller bucket with water
    3. Place the aquarium pump (with tubes attached to the Peltier heat exchanger) into the bucket and plug in the pump so that water flows
    4. Turn on the peltier controller (switch in the back of the instrument)
    5. Use the arrow buttons to set the temperature.
  2. Turning off the Peltier controller
    1. Turn the controller power off
    2. Unplug the aquarium pump
    3. Dump the water from the bucket into the sink

Methods

  1. Sample stock solutions were prepared.
  2. The fluorescence protocol above was followed to operate the instrument
  3. A fluorescence scan was taken every five minutes for 165 minutes.
  4. Data were compiled using the command tab on the lab computer and the .jar file.
  5. The data were analyzed.

Fluorescence Samples

Stocks

  1. Distilled Water
  2. 5.08 mM Au
  3. 37.93 mM BSA
  4. 1 mM HCl

BSA Fluorescence

  1. 3 mL total
  2. 0.25 mM Au
  3. 3.125 μM BSA
  4. HCl or NaOH appropriate for your pH (4) water

Solution:

  1. 2306 uL HOH
  2. 147 uL AuCl3
  3. 247 uL BSA
  4. 300 uL HCl

Fluorescence Data

The sample was placed into the fluorometer and a scan was taken every five minutes after being blanked. The fluorescence summary shows the fluorescence scans at selected timepoints. By inspection of the chart, it is apparent that the peak increases over time. The peak around 325 nm can be attributed to water Raman scattering.

Image:2. Fluor summary AMF 16.PNG

The fluorescence peak does not increase dramatically after 40 minutes. This chart is an abridged version of the above chart, showing only sample runs under 40 minutes. The bulk of the fluorescence intensity increase occurs between (0-5) and (5-10) minutes.

Image:3. Fluor summary abridged AMF 16.PNG

The emission maximum over time is shown in the chart below. The most dramatic shift in emission maximums occurs between 5 and 10 minutes.

Image:1. Emission AMF Flour.PNG

The integrated intensity over time is shown below. The intensity dramatically increases until 25 minutes (peak) at which point it slowly falls until about 65 minutes. It then slowly increases until the end of the run. The peak at 25 minutes shows the full unfolding of the protein, allowing both tryptophan residues to fluoresce.

Image:5 Integrated Intensity Full summary AMF 16.PNG

This chart shows an abridged version of the above chart in order to show the increase in integrated intensity (and unfolding of BSA) in the first 40 minutes.

Image:4 Integrated intensity abridged AMF 16.PNG

Preparation of UV-Vis samples for 160913

BSA for UV-Vis during next session

  1. 5 mL total
  2. 0.25 mM Au
  3. 3.125 μM BSA
  4. HCl or NaOH appropriate for your pH
  5. Appropriate amount of fructose
  6. water


stock solutions

  1. The NaOH and HCl samples were made exactly as designed.
  2. AuCl3
    1. mass = 38.5 mg
    2. 25.0 mL volumetric flask
    3. concentration = 5.08 mM
  3. Fructose
    1. mass = 23.48 mg
    2. 10.0 mL volumetric flask
    3. concentration = 13.0 mM
  4. Bovine Serum Albumin
    1. mass = 25.33 mg
    2. 10.0 mL volumetric flask
    3. concentration = 37.93 mM
0.125 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of BSAstock μL of Water
4 500 0 0 246 48 408 3798
5 50 0 0 246 48 408 4248
6 5 0 0 246 48 408 4293
7 0 0 0 246 48 408 4298
8 0 5 0 246 48 408 4293
9 0 50 0 246 48 408 4248
10 0 500 0 246 48 408 3798
11 0 0 5 246 48 408 4293
12 0 0 50 246 48 408 4248
0.25 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of BSAstock μL of Water
4 500 0 0 246 96 408 3750
5 50 0 0 246 96 408 4200
6 5 0 0 246 96 408 4245
7 0 0 0 246 96 408 4250
8 0 5 0 246 96 408 4245
9 0 50 0 246 96 408 4200
10 0 500 0 246 96 408 3570
11 0 0 5 246 96 408 4245
12 0 0 50 246 96 408 4200


Solutions before being heated: Image:160907 stock solutions 671 lab.jpg

Solutions were added to glass test tubes, covered with aluminum foil, and placed in an oven (set to 80°C) for 4 hours.

  • Matt Hartings 14:01, 9 September 2016 (EDT): I remade your samples because you got strange results. Here's my description


1909

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