Make stock solutions of lysozyme and determine its molar absorptivity using the UV-Vis and Fluorescence Spectrophotometers
Determine how protein concentration affects fluorescence intensity
Prep a sample 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
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:
For the 50.88µM solution:
We measured out 7.28mg of the lysozyme on the milligram balance using a weigh boat
We used HPLC water to rinse the lysozyme into a clean 10mL volumetric flask
We filled the volumetric flask to the 10mL line using HPLC water
We transferred the sample into a new 15mL falcon tube
For the remaining solutions:
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.
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.
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.
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.
Then, we prepped our stock samples of our protease, alpha-chymotrypsin.
First, we weighed the mass of the 1.5mL Eppindorf tube that we were going to use to store our stock of alpha-chymotrypsin.
Next, we added approximately 1mg of dry alpha-chymotrypsin to the Eppindorf tube.
We then closed the tube and labeled it with the following:
The date (20150909)
The molarity that the stock of alpha-chemotrypsin would be upon addition of 1mL HPLC water
Alpha-chymotrypsin
Our group's initials (BJN)
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.
After that, we took absorbance data for each of our lysozyme samples using UV-Vis Spectroscopy.
Data and Measurements
alpha-Chymotrpysin Standards
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)
The figure above shows the absorbance of each lysozyme standard as a function of the wavelength of incident light. The absorbance values were corrected for the water blank and dark noise.
Table 2: Absorbance of Blank (HPLC Water) and Each Lysozyme Standard at a Wavelength of 280nm
The table above shows the absorbance of each lysozyme standard at a wavelength of 280nm. This data is shown graphically in the figure below.
Figure 2: Absorbance of Each Lysozyme Standard as a Function of Concentration (µM) at a Wavelength of 280nm
The figure above shows the absorbance of lysozyme as a function of its concentration at a wavelength of incident light of 280nm. A trend line was added and set to a y-intercept of 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 0.0337. This represents the molar absorptivity. Thus, the molar absorptivity was 0.0337µM-1cm-1, or 33.7mol-1cm-1.
Fluorescence
Figure 3: Fluorescence as a Function of Wavelength for Each Lysozyme Standard
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)
Figure 4: Intensity of Re-emitted Light as a Function of Concentration (M)
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?
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