User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/09

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Objective

The purpose of today's work is to:

  1. Make stock solutions of lysozyme
  2. Measure fluorescence intensity and absorbance of lysozyme as a function of the concentration of lysozyme
  3. Determine the molar absorptivity of lysozyme using the UV-Vis and Fluorescence Spectrophotometers
  4. Determine how protein concentration affects fluorescence intensity
  5. Prep samples of our protease, alpha-chymotrypsin, for use in future experiments

A full description of today's objectives and protocols are in Dr. Hartings's lab notebook.

Procedure

  1. Frist, we made our stock solutions of lysozyme. We made solutions of 50.88µM, 15.445µM, 7.7225µM, 3.86µM, 1.93µM, and 0.96µM lysozyme in HPLC water as follows:
    1. For the 50.88µM solution:
      1. We measured out 7.28mg of the lysozyme on the milligram balance using a weigh boat
      2. We used HPLC water to rinse the lysozyme into a clean 10mL volumetric flask
      3. We filled the volumetric flask to the 10mL line using HPLC water
      4. We transferred the sample into a new 15mL falcon tube
    2. For the remaining solutions:
      1. We weighed out 4.42mg of the lysozyme in a weigh boat using the milligram balance and diluted the lysozyme in HPLC water, using a volumetric flask, to a total volume of 10mL. This solution had a molarity of 30.89µM.
      2. For our first stock solution, we pipetted 5mL of the 30.89µM lysozyme solution into a new 15mL falcon tube and then pipetted 5mL of HPLC into the tube to bring the final molarity to 15.445µM. We labeled the tube in green tape with the concentration.
      3. For our second stock solution, we pipetted 5mL of the 15.445µM solution into a new 15mL falcon tube and then pipetted 5mL of HPLC into the tube to bring the final molarity to 7.7225µM. We labeled the tube in green tape with the concentration.
      4. We continued doing serial dilutions in this way to make three more samples, one of each with a molarity of 3.86µM, 1.93µM, and 0.96µM.
  2. Then, we prepped our stock samples of our protease, alpha-chymotrypsin, for use in future experiments.
    1. First, we weighed the mass of the 1.5mL Eppindorf tube that we were going to use to store our stock of alpha-chymotrypsin.
    2. Next, we added approximately 1mg of dry alpha-chymotrypsin to the Eppindorf tube.
    3. We then closed the tube and labeled it with the following:
      1. The date (20150909)
      2. The molarity that the stock of alpha-chemotrypsin would be upon addition of 1mL HPLC water
      3. Alpha-chymotrypsin
      4. Our group's initials (BJN)
    4. We did this 15 times. All tubes were stored in a cardboard cryo box with our initials on it in the freezer. A table that shows the mass of the Eppindorf tube, the mass of the alpha-chymotrypsin, and the molarity that the stock would be upon addition of 1mL water is in the Data and Measurements section.
  3. After that, we took absorbance and fluorescence data for each of our lysozyme samples.

Data and Measurements

Table 1: Measurements for Each of the 15 alpha-Chymotrpysin Standards: Mass of Eppindorf Tube (g), Mass of alpha-Chymotrypsin (g), and Molarity of alpha-Chymotrypsin in 1mL HPLC Water (M)


Mass of Eppindorf Tube (g) Mass of alpha-Chymotrypsin (g) Molarity (µM)
1.1017 0.00122 47.656
1.015 0.00137 53.515625
1.01348 0.00131 51.171875
1.0179 0.00105 41.015625
1.02954 0.00135 52.734375
1.0228 0.00102 39.84375
1.02048 0.0013 50.78125
1.01945 0.00111 43.359375
1.02723 0.00099 38.671875
1.02951 0.00105 41.015625
1.01517 0.00108 42.1875
1.02954 0.00094 36.71875
1.03211 0.00104 40.625
1.0192 0.00111 43.359375
1.02781 0.00121 47.265625

Analysis

UV-Vis

Figure 1: Absorbance of Each Lysozyme Standard as a Function of Wavelength of Incident Light (nm)


20151103 bonan corrected absorbance from 0909.png

The figure above shows the absorbance of each lysozyme standard as a function of the wavelength of incident light. The absorbance values were first corrected for the water blank by subtracting the absorbance of the blank at each wavelength from that of the samples. The absorbance was then corrected for instrumental noise by subtracting the average of the last 100nm from every data point.


Figure 2: Absorbance of Each Lysozyme Standard as a Function of Concentration (µM) at a Wavelength of 280nm


20151103 bonan corrected calib curve from 0909.png

The figure above shows the absorbance of lysozyme as a function of its concentration at a wavelength of incident light of 280nm. A linear trend line was added with the y-intercept set to 0. The equation for this trend line, along with its r squared value, is shown on the graph.

The slope of the line of best fit is 2364.1. This represents the molar absorptivity. Thus, the molar absorptivity was 2364.1µM-1cm-1, or 23.6mol-1cm-1.

Fluorescence

Figure 3: Fluorescence as a Function of Wavelength for Each Lysozyme Standard


20150909 bonan fluorescence graph.png

The values for fluorescence were corrected for the water blank.

The area under the fluorescence curve was integrated for each lysozyme standard, giving the intensity of the the fluorescence for each sample. A table and a plot of the intensity as a function of concentration are below.


Table 3: Intensity of Re-emitted Light from Each Lysozyme Sample as a Function of Concentration (M)


20150909 bonan intensity conc chart.png


Figure 4: Intensity of Re-emitted Light as a Function of Concentration (M)


20150909 bonan intensity conc.png

Matt Hartings Lots of data here! You don't have to use the raw data for your spectra. Just show your graphs. In your A vs conc graph, show your data as points only. Keep the line from the fit. What is your measured molar absorptivity M-1 cm-1?