User:Benjamin Friedel/Notebook/CHEM 471/2015/09/09

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UV-Vis and Fluorescence Analysis of Lysozyme and Protease Sample Prep

Objective

There are two objectives for 09/02/2015. First, to calculate the molar absorptivity of lysozyme through UV-Vis and Fluorescence spectroscopy. Second, to prepare protease samples (using Chymotrypsin) for use in experiments at a later date.

Procedure and Data

1) First, Lysozyme samples were prepared for spectral analysis. A 10mL solution of 50.88uM lysozyme (MW=14307g/mol) in HPLC grade water was made by dissolving 7.28mg lysozyme in 10ml HPLC grade water.

    1. Solution was measured in a volumetric flask after lysozyme mass was measured on milligram balance in weigh boat.
    2. Solution stored in 15mL falcon tube.
  1. Further Lysozyme Solutions were made in concentrations of 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM
    1. First, 10mL of a 30.89uM lysozyme solution in HPLC grade water was made by adding 4.42mg lysozyme in 10ml water in a 10ml volumetric flask.
    2. Then, serial dilutions were carried out using a 5ml pipette, dividing concentration of original solution by 1/2 each dilution by removing 5mL of the solution and adding it to 5mL of HPLC grade water.This was carried out 5 times to reach the above lysozyme concentrations, achieving 5mL of each solution except for the 0.965uM solution of which there were 10mL.
      1. ex.) For the 15.445uM solution, 5mL of the 30.89uM solution were pipetted into a 15mL falcon tube containing 5mL of HPLC grade water.
    3. Solution were stored at room temperature in 15mL falcon tubes and labeled with green tape.


2) UV-Vis Data was then collected for the 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM lysozyme samples as well as an HPLC water blank from 200nm-800nm.

    1. First a water blank was measured
    2. Then the spectrum of each of the above noted samples was measured
    3. The spectra for each sample was corrected for solvent and baseline
      1. For blank correction, the blank spectra was subtracted from each sample spectra according to wavelength
      2. for baseline correction, the average absorbance was calculated between 700-800nm (lowest activity ranges as protein absorbs most highly at 280nm) for each sample spectra individually and then subtracted from that respective spectra.

Table 1: UV-Vis Raw and Adjusted Data from 200nm-800nm

' HPLC water blank 0.956uM 0.956uM-blank 0.956uM-blank-baseline 1.93uM 1.93uM-blank 1.93uM-blank-baseline 3.86uM 3.86uM-blank 3.86uM-blank-baseline 7.225uM 7.225uM-blank 7.225uM-blank-baseline 15.445uM 15.445uM-blank 15.445uM-blank-baseline
Wavelength(nm)Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.Abs.
2000.0910.6860.5950.559237621.3051.2141.1472.5562.4652.3874.7984.7074.63954.9094.84
2010.0840.6340.550.514237621.2031.1191.0522.3562.2722.1944.7984.7144.64654.9164.847
2020.080.5820.5020.466237621.1041.0240.9572.1652.0852.0074.0233.9433.87554.924.851
2030.0750.5360.4610.425237621.0160.9410.8741.9871.9121.8343.8983.8233.75554.9254.856
2040.0710.4950.4240.388237620.940.8690.8021.8321.7611.6833.5733.5023.43454.9294.86
2050.0680.4590.3910.355237620.8740.8060.7391.6981.631.5523.2733.2053.13754.9324.863
2060.0650.4280.3630.327237620.8170.7520.6851.581.5151.4373.0562.9912.92354.9354.866
2070.0620.40.3380.302237620.7630.7010.6341.4741.4121.3342.8462.7842.71654.9384.869
2080.060.3760.3160.280237620.7170.6570.591.3841.3241.2462.672.612.54254.944.871
2090.0570.3550.2980.262237620.6790.6220.5551.3061.2491.1712.5322.4752.40754.9434.874
2100.0560.3390.2830.247237620.6470.5910.5241.2461.191.1122.4092.3532.28554.9444.875
2110.0540.3250.2710.235237620.620.5660.4991.1921.1381.062.3062.2522.18454.9464.877
2120.0520.3130.2610.225237620.5970.5450.4781.1481.0961.0182.2192.1672.0994.4984.4464.377
2130.0520.3040.2520.216237620.5780.5260.4591.1111.0590.9812.1492.0972.0294.4984.4464.377
2140.0510.2970.2460.210237620.5640.5130.4461.0841.0330.9552.0972.0461.9784.4984.4474.378
2150.050.290.240.204237620.5520.5020.4351.0621.0120.9342.0562.0061.9384.1984.1484.079
2160.0490.2860.2370.201237620.5440.4950.4281.0440.9950.9172.0231.9741.9064.1984.1494.08
2170.0480.2830.2350.199237620.5360.4880.4211.0310.9830.9051.9961.9481.884.1024.0543.985
2180.0470.280.2330.197237620.5290.4820.4151.0180.9710.8931.9751.9281.864.1024.0553.986
2190.0460.2760.230.194237620.5220.4760.4091.0060.960.8821.951.9041.8363.9563.913.841
2200.0460.2730.2270.191237620.5150.4690.4020.990.9440.8661.9221.8761.8083.8483.8023.733
2210.0460.2680.2220.186237620.5050.4590.3920.9720.9260.8481.8851.8391.7713.7613.7153.646
2220.0450.2620.2170.181237620.4920.4470.380.9470.9020.8241.8351.791.7223.6563.6113.542
2230.0450.2540.2090.173237620.4770.4320.3650.9170.8720.7941.7771.7321.6643.5023.4573.388
2240.0450.2460.2010.165237620.4590.4140.3470.8820.8370.7591.7041.6591.5913.3723.3273.258
2250.0440.2360.1920.156237620.4390.3950.3280.8430.7990.7211.6261.5821.5143.2033.1593.09
2260.0430.2240.1810.145237620.4160.3730.3060.7950.7520.6741.5311.4881.423.0052.9622.893
2270.0430.2110.1680.132237620.390.3470.280.7440.7010.6231.4291.3861.3182.7842.7412.672
2280.0420.1970.1550.119237620.3620.320.2530.6860.6440.5661.3151.2731.2052.5562.5142.445
2290.0420.1810.1390.103237620.3310.2890.2220.6220.580.5021.1891.1471.0792.32.2582.189
2300.0410.1640.1230.087237620.2980.2570.190.5570.5160.4381.0591.0180.952.0381.9971.928
2310.040.1470.1070.071237620.2650.2250.1580.4890.4490.3710.9260.8860.8181.7761.7361.667
2320.0390.1310.0920.056237620.2350.1960.1290.4270.3880.310.8060.7670.6991.5391.51.431
2330.0380.1170.0790.043237620.2060.1680.1010.370.3320.2540.6940.6560.5881.3251.2871.218
2340.0380.1040.0660.030237620.180.1420.0750.3180.280.2020.5930.5550.4871.131.0921.023
2350.0370.0930.0560.020237620.1590.1220.0550.2760.2390.1610.5120.4750.4070.9720.9350.866
2360.0360.0830.0470.011237620.1410.1050.0380.2410.2050.1270.4430.4070.3390.8420.8060.737
2370.0360.0770.0410.005237620.1270.0910.0240.2130.1770.0990.390.3540.2860.7380.7020.633
2380.0350.0710.0360.000237620.1150.080.0130.1910.1560.0780.3450.310.2420.650.6150.546
2390.0350.0650.03-0.005762380.1050.070.0030.1710.1360.0580.3060.2710.2030.5740.5390.47
2400.0350.0610.026-0.009762380.0970.062-0.0050.1550.120.0420.2750.240.1720.5130.4780.409
2410.0340.0580.024-0.011762380.090.056-0.0110.1410.1070.0290.2490.2150.1470.4610.4270.358
2420.0340.0550.021-0.014762380.0850.051-0.0160.130.0960.0180.2270.1930.1250.4160.3820.313
2430.0330.0530.02-0.015762380.080.047-0.020.120.0870.0090.2090.1760.1080.3810.3480.279
2440.0330.050.017-0.018762380.0760.043-0.0240.1130.080.0020.1940.1610.0930.3510.3180.249
2450.0330.0490.016-0.019762380.0730.04-0.0270.1070.074-0.0040.1820.1490.0810.3280.2950.226
2460.0320.0470.015-0.020762380.070.038-0.0290.1020.07-0.0080.1720.140.0720.310.2780.209
2470.0320.0460.014-0.021762380.0680.036-0.0310.0970.065-0.0130.1650.1330.0650.2950.2630.194
2480.0320.0450.013-0.022762380.0670.035-0.0320.0950.063-0.0150.1590.1270.0590.2850.2530.184
2490.0320.0450.013-0.022762380.0660.034-0.0330.0930.061-0.0170.1550.1230.0550.2780.2460.177
2500.0320.0440.012-0.023762380.0650.033-0.0340.0910.059-0.0190.1530.1210.0530.2740.2420.173
2510.0310.0440.013-0.022762380.0640.033-0.0340.0910.06-0.0180.1520.1210.0530.2730.2420.173
2520.0310.0430.012-0.023762380.0640.033-0.0340.0910.06-0.0180.1530.1220.0540.2740.2430.174
2530.0310.0430.012-0.023762380.0640.033-0.0340.0910.06-0.0180.1540.1230.0550.2770.2460.177
2540.030.0440.014-0.021762380.0650.035-0.0320.0930.063-0.0150.1570.1270.0590.2820.2520.183
2550.030.0440.014-0.021762380.0650.035-0.0320.0950.065-0.0130.1610.1310.0630.2890.2590.19
2560.030.0440.014-0.021762380.0660.036-0.0310.0970.067-0.0110.1650.1350.0670.2980.2680.199
2570.030.0450.015-0.020762380.0680.038-0.0290.10.07-0.0080.170.140.0720.3090.2790.21
2580.030.0460.016-0.019762380.0680.038-0.0290.1020.072-0.0060.1760.1460.0780.320.290.221
2590.030.0460.016-0.019762380.070.04-0.0270.1050.075-0.0030.1820.1520.0840.3320.3020.233
2600.030.0460.016-0.019762380.0710.041-0.0260.1080.07800.1880.1580.090.3440.3140.245
2610.030.0470.017-0.018762380.0720.042-0.0250.1110.0810.0030.1950.1650.0970.3570.3270.258
2620.030.0480.018-0.017762380.0740.044-0.0230.1150.0850.0070.2020.1720.1040.3710.3410.272
2630.0290.0490.02-0.015762380.0760.047-0.020.1180.0890.0110.210.1810.1130.3860.3570.288
2640.0290.0490.02-0.015762380.0770.048-0.0190.1220.0930.0150.2160.1870.1190.4010.3720.303
2650.0290.050.021-0.014762380.0790.05-0.0170.1250.0960.0180.2240.1950.1270.4150.3860.317
2660.0290.0510.022-0.013762380.0810.052-0.0150.1290.10.0220.2310.2020.1340.430.4010.332
2670.0290.0510.022-0.013762380.0820.053-0.0140.1330.1040.0260.2390.210.1420.4450.4160.347
2680.0290.0520.023-0.012762380.0840.055-0.0120.1360.1070.0290.2460.2170.1490.4610.4320.363
2690.0290.0530.024-0.011762380.0860.057-0.010.140.1110.0330.2530.2240.1560.4740.4450.376
2700.0290.0540.025-0.010762380.0880.059-0.0080.1430.1140.0360.260.2310.1630.4880.4590.39
2710.0280.0540.026-0.009762380.0890.061-0.0060.1460.1180.040.2650.2370.1690.4990.4710.402
2720.0280.0540.026-0.009762380.0890.061-0.0060.1480.120.0420.270.2420.1740.5090.4810.412
2730.0290.0550.026-0.009762380.090.061-0.0060.150.1210.0430.2750.2460.1780.5170.4880.419
2740.0280.0560.028-0.007762380.0910.063-0.0040.1510.1230.0450.2770.2490.1810.5240.4960.427
2750.0280.0560.028-0.007762380.0910.063-0.0040.1520.1240.0460.2790.2510.1830.5280.50.431
2760.0280.0550.027-0.008762380.0910.063-0.0040.1520.1240.0460.280.2520.1840.530.5020.433
2770.0280.0550.027-0.008762380.0910.063-0.0040.1530.1250.0470.2810.2530.1850.5310.5030.434
2780.0280.0560.028-0.007762380.0920.064-0.0030.1540.1260.0480.2830.2550.1870.5350.5070.438
2790.0280.0560.028-0.007762380.0930.065-0.0020.1550.1270.0490.2850.2570.1890.5390.5110.442
2800.0280.0560.028-0.007762380.0930.065-0.0020.1560.1280.050.2870.2590.1910.5430.5150.446
2810.0270.0560.029-0.006762380.0930.066-0.0010.1560.1290.0510.2870.260.1920.5440.5170.448
2820.0270.0560.029-0.006762380.0920.065-0.0020.1550.1280.050.2860.2590.1910.5410.5140.445
2830.0280.0550.027-0.008762380.0910.063-0.0040.1530.1250.0470.2820.2540.1860.5330.5050.436
2840.0280.0540.026-0.009762380.0890.061-0.0060.1490.1210.0430.2740.2460.1780.5180.490.421
2850.0270.0530.026-0.009762380.0860.059-0.0080.1430.1160.0380.2630.2360.1680.4950.4680.399
2860.0270.0510.024-0.011762380.0830.056-0.0110.1360.1090.0310.250.2230.1550.4690.4420.373
2870.0270.050.023-0.012762380.080.053-0.0140.1310.1040.0260.2380.2110.1430.4430.4160.347
2880.0280.0490.021-0.014762380.0790.051-0.0160.1270.0990.0210.230.2020.1340.4280.40.331
2890.0270.0490.022-0.013762380.0790.052-0.0150.1260.0990.0210.2280.2010.1330.4240.3970.328
2900.0270.0480.021-0.014762380.0780.051-0.0160.1250.0980.020.2270.20.1320.4220.3950.326
2910.0270.0480.021-0.014762380.0770.05-0.0170.1230.0960.0180.2220.1950.1270.4110.3840.315
2920.0270.0460.019-0.016762380.0730.046-0.0210.1150.0880.010.2070.180.1120.3830.3560.287
2930.0270.0430.016-0.019762380.0670.04-0.0270.1040.077-0.0010.1840.1570.0890.3370.310.241
2940.0270.040.013-0.022762380.0610.034-0.0330.0920.065-0.0130.1590.1320.0640.2880.2610.192
2950.0270.0370.01-0.025762380.0560.029-0.0380.0790.052-0.0260.1350.1080.040.2410.2140.145
2960.0270.0340.007-0.028762380.0510.024-0.0430.0690.042-0.0360.1150.0880.020.2020.1750.106
2970.0270.0320.005-0.030762380.0470.02-0.0470.0610.034-0.0440.0980.0710.0030.1690.1420.073
2980.0270.0310.004-0.031762380.0430.016-0.0510.0540.027-0.0510.0840.057-0.0110.1420.1150.046
2990.0260.0290.003-0.032762380.0410.015-0.0520.0480.022-0.0560.0730.047-0.0210.1180.0920.023
3000.0260.0280.002-0.033762380.0390.013-0.0540.0440.018-0.060.0630.037-0.0310.0990.0730.004
3010.0270.0280.001-0.034762380.0370.01-0.0570.040.013-0.0650.0560.029-0.0390.0840.057-0.012
3020.0270.0270-0.035762380.0350.008-0.0590.0370.01-0.0680.0490.022-0.0460.070.043-0.026
3030.0270.026-0.001-0.036762380.0340.007-0.060.0350.008-0.070.0450.018-0.050.0610.034-0.035
3040.0270.026-0.001-0.036762380.0330.006-0.0610.0330.006-0.0720.0410.014-0.0540.0540.027-0.042
3050.0270.026-0.001-0.036762380.0330.006-0.0610.0310.004-0.0740.0380.011-0.0570.0490.022-0.047
3060.0260.025-0.001-0.036762380.0310.005-0.0620.030.004-0.0740.0360.01-0.0580.0440.018-0.051
3070.0270.025-0.002-0.037762380.0310.004-0.0630.030.003-0.0750.0340.007-0.0610.0410.014-0.055
3080.0270.025-0.002-0.037762380.0310.004-0.0630.0290.002-0.0760.0330.006-0.0620.0390.012-0.057
3090.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.0320.005-0.0630.0380.011-0.058
3100.0270.024-0.003-0.038762380.0310.004-0.0630.0280.001-0.0770.0320.005-0.0630.0360.009-0.06
3110.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.0320.005-0.0630.0350.008-0.061
3120.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.0310.004-0.0640.0350.008-0.061
3130.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.0310.004-0.0640.0350.008-0.061
3140.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.030.003-0.0650.0340.007-0.062
3150.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.030.003-0.0650.0340.007-0.062
3160.0270.025-0.002-0.037762380.030.003-0.0640.0280.001-0.0770.030.003-0.0650.0340.007-0.062
3170.0270.024-0.003-0.038762380.030.003-0.0640.0280.001-0.0770.030.003-0.0650.0340.007-0.062
3180.0280.024-0.004-0.039762380.030.002-0.0650.0280-0.0780.0310.003-0.0650.0340.006-0.063
3190.0270.025-0.002-0.037762380.030.003-0.0640.0280.001-0.0770.030.003-0.0650.0340.007-0.062
3200.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0330.007-0.062
3210.0260.023-0.003-0.038762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0330.007-0.062
3220.0260.024-0.002-0.037762380.0290.003-0.0640.0260-0.0780.0290.003-0.0650.0320.006-0.063
3230.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.030.004-0.0640.0330.007-0.062
3240.0270.024-0.003-0.038762380.030.003-0.0640.0270-0.0780.0290.002-0.0660.0330.006-0.063
3250.0270.024-0.003-0.038762380.0290.002-0.0650.0270-0.0780.0290.002-0.0660.0320.005-0.064
3260.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0320.006-0.063
3270.0270.024-0.003-0.038762380.030.003-0.0640.0270-0.0780.0290.002-0.0660.0330.006-0.063
3280.0270.024-0.003-0.038762380.0290.002-0.0650.0280.001-0.0770.0290.002-0.0660.0330.006-0.063
3290.0260.024-0.002-0.037762380.0290.003-0.0640.0280.002-0.0760.0290.003-0.0650.0320.006-0.063
3300.0270.024-0.003-0.038762380.0290.002-0.0650.0270-0.0780.0290.002-0.0660.0320.005-0.064
3310.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0320.006-0.063
3320.0270.024-0.003-0.038762380.0290.002-0.0650.0270-0.0780.0290.002-0.0660.0320.005-0.064
3330.0270.024-0.003-0.038762380.030.003-0.0640.0270-0.0780.0290.002-0.0660.0320.005-0.064
3340.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0310.005-0.064
3350.0260.024-0.002-0.037762380.0290.003-0.0640.0270.001-0.0770.0290.003-0.0650.0310.005-0.064
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7390.0420.038-0.004-0.039762380.04-0.002-0.0690.0440.002-0.0760.041-0.001-0.0690.041-0.001-0.07
7400.0420.038-0.004-0.039762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7410.0420.038-0.004-0.039762380.04-0.002-0.0690.0440.002-0.0760.041-0.001-0.0690.041-0.001-0.07
7420.0420.038-0.004-0.039762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7430.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7440.0420.038-0.004-0.039762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7450.0420.038-0.004-0.039762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7460.0420.039-0.003-0.038762380.04-0.002-0.0690.0440.002-0.0760.041-0.001-0.0690.041-0.001-0.07
7470.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7480.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7490.0420.038-0.004-0.039762380.041-0.001-0.0680.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7500.0430.039-0.004-0.039762380.04-0.003-0.070.0450.002-0.0760.041-0.002-0.070.041-0.002-0.071
7510.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7520.0420.039-0.003-0.038762380.04-0.002-0.0690.0440.002-0.0760.041-0.001-0.0690.041-0.001-0.07
7530.0430.039-0.004-0.039762380.041-0.002-0.0690.0460.003-0.0750.042-0.001-0.0690.041-0.002-0.071
7540.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.0420-0.0680.041-0.001-0.07
7550.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.041-0.001-0.0690.041-0.001-0.07
7560.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.041-0.001-0.0690.041-0.001-0.07
7570.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7580.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.0420-0.0680.0420-0.069
7590.0420.039-0.003-0.038762380.041-0.001-0.0680.0460.004-0.0740.0420-0.0680.041-0.001-0.07
7600.0430.039-0.004-0.039762380.04-0.003-0.070.0460.003-0.0750.041-0.002-0.070.041-0.002-0.071
7610.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.041-0.001-0.0690.041-0.001-0.07
7620.0430.039-0.004-0.039762380.04-0.003-0.070.0450.002-0.0760.042-0.001-0.0690.041-0.002-0.071
7630.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7640.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.041-0.001-0.0690.041-0.001-0.07
7650.0430.039-0.004-0.039762380.041-0.002-0.0690.0460.003-0.0750.042-0.001-0.0690.041-0.002-0.071
7660.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.0420-0.0680.041-0.001-0.07
7670.0420.039-0.003-0.038762380.04-0.002-0.0690.0450.003-0.0750.041-0.001-0.0690.041-0.001-0.07
7680.0430.039-0.004-0.039762380.04-0.003-0.070.0460.003-0.0750.042-0.001-0.0690.041-0.002-0.071
7690.0430.039-0.004-0.039762380.04-0.003-0.070.0470.004-0.0740.041-0.002-0.070.041-0.002-0.071
7700.0420.039-0.003-0.038762380.04-0.002-0.0690.0460.004-0.0740.041-0.001-0.0690.041-0.001-0.07
7710.0430.038-0.005-0.040762380.04-0.003-0.070.0460.003-0.0750.042-0.001-0.0690.041-0.002-0.071
7720.0420.039-0.003-0.038762380.039-0.003-0.070.0460.004-0.0740.041-0.001-0.0690.04-0.002-0.071
7730.0420.039-0.003-0.038762380.039-0.003-0.070.0460.004-0.0740.041-0.001-0.0690.04-0.002-0.071
7740.0420.038-0.004-0.039762380.04-0.002-0.0690.0470.005-0.0730.041-0.001-0.0690.04-0.002-0.071
7750.0420.038-0.004-0.039762380.039-0.003-0.070.0460.004-0.0740.041-0.001-0.0690.04-0.002-0.071
7760.0420.038-0.004-0.039762380.039-0.003-0.070.0450.003-0.0750.041-0.001-0.0690.04-0.002-0.071
7770.0430.039-0.004-0.039762380.04-0.003-0.070.0470.004-0.0740.041-0.002-0.070.04-0.003-0.072
7780.0420.039-0.003-0.038762380.039-0.003-0.070.0460.004-0.0740.041-0.001-0.0690.04-0.002-0.071
7790.0420.038-0.004-0.039762380.039-0.003-0.070.0460.004-0.0740.041-0.001-0.0690.04-0.002-0.071
7800.0420.038-0.004-0.039762380.039-0.003-0.070.0460.004-0.0740.04-0.002-0.070.04-0.002-0.071
7810.0420.038-0.004-0.039762380.039-0.003-0.070.0450.003-0.0750.041-0.001-0.0690.039-0.003-0.072
7820.0410.037-0.004-0.039762380.038-0.003-0.070.0450.004-0.0740.04-0.001-0.0690.04-0.001-0.07
7830.0420.038-0.004-0.039762380.038-0.004-0.0710.0450.003-0.0750.04-0.002-0.070.04-0.002-0.071
7840.0410.038-0.003-0.038762380.038-0.003-0.070.0450.004-0.0740.04-0.001-0.0690.039-0.002-0.071
7850.0410.038-0.003-0.038762380.038-0.003-0.070.0460.005-0.0730.04-0.001-0.0690.039-0.002-0.071
7860.0410.037-0.004-0.039762380.038-0.003-0.070.0450.004-0.0740.039-0.002-0.070.039-0.002-0.071
7870.0410.037-0.004-0.039762380.038-0.003-0.070.0460.005-0.0730.039-0.002-0.070.039-0.002-0.071
7880.0410.038-0.003-0.038762380.038-0.003-0.070.0460.005-0.0730.04-0.001-0.0690.04-0.001-0.07
7890.0410.038-0.003-0.038762380.038-0.003-0.070.0450.004-0.0740.039-0.002-0.070.039-0.002-0.071
7900.0410.037-0.004-0.039762380.038-0.003-0.070.0460.005-0.0730.039-0.002-0.070.038-0.003-0.072
7910.0410.037-0.004-0.039762380.038-0.003-0.070.0460.005-0.0730.039-0.002-0.070.038-0.003-0.072
7920.0410.038-0.003-0.038762380.038-0.003-0.070.0460.005-0.0730.039-0.002-0.070.039-0.002-0.071
7930.040.037-0.003-0.038762380.037-0.003-0.070.0450.005-0.0730.039-0.001-0.0690.038-0.002-0.071
7940.040.037-0.003-0.038762380.037-0.003-0.070.0440.004-0.0740.039-0.001-0.0690.038-0.002-0.071
7950.0390.036-0.003-0.038762380.037-0.002-0.0690.0440.005-0.0730.038-0.001-0.0690.037-0.002-0.071
7960.040.036-0.004-0.039762380.037-0.003-0.070.0430.003-0.0750.039-0.001-0.0690.038-0.002-0.071
7970.040.036-0.004-0.039762380.037-0.003-0.070.0450.005-0.0730.038-0.002-0.070.038-0.002-0.071
7980.0390.036-0.003-0.038762380.037-0.002-0.0690.0430.004-0.0740.038-0.001-0.0690.038-0.001-0.07
7990.040.036-0.004-0.039762380.037-0.003-0.070.0450.005-0.0730.038-0.002-0.070.038-0.002-0.071
8000.040.036-0.004-0.039762380.037-0.003-0.070.0450.005-0.0730.038-0.002-0.070.038-0.002-0.071


Table 2: Absorbance of Each Lysozyme Concentration Adjusted for HPLC Water Blank and Baseline at 280nm

Concentration ( uM) Absorption
0.956-0.007762376
1.93-0.002
3.860.05
7.2250.191
15.4450.446

Figure 1: Lysozyme Absorption at 280nm as a Function of Concentration


Description of image

From this we can see that our molar absorptivity for lysozyme would be .0325Lumol-1cm-1 or 32.5mol-1cm-1 as A-Ebc, with E being the slope as b=1.

3) Fluorescence data was then measured for the 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM lysozyme samples as well as an HPLC water blank.

    1. First a water blank was measured
    2. Then the spectrum of each of the above noted samples was measured
    3. The spectra for each sample was corrected for solvent blank
      1. For blank correction, the blank spectra was subtracted from each sample spectra according to wavelength

Table 3: Fluorescence Raw and Adjusted Data from 300nm-500nm Wavelengths

Wavelength (nm) HPLC Water blank 0.965uM 0.965-blank 1.93uM 1.93-blank 3.86uM 3.86-blank 7.225uM 7.225-blank 15.445uM 15.445-blank
Emission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission IntensityEmission Intensity
3000.2411.7141.4734.0443.8037.2236.98210.019.76914.05313.812
300.50.2317982.033251.8014524.3195384.087747.7360197.50422111.30419111.07239315.80322215.571424
3010.2252612.3388882.1136274.6145354.3892748.2854358.06017412.59032212.36506117.54744217.322181
301.50.2230472.6350712.4120244.9493614.7263148.9033018.68025413.88991313.66686619.31529319.092246
3020.2285522.9249922.696445.3237785.0952269.5921619.36360915.22566114.99710921.12950920.900957
302.50.2445223.2127842.9682625.7397725.4952510.37519210.1306716.60324816.35872623.00891422.764392
3030.2672493.4981363.2308876.2132065.94595711.25008810.98283918.02972917.7624824.98109624.713847
303.50.295493.7823483.4868586.7438376.44834712.22167111.92618119.51579219.22030227.0787626.78327
3040.3305134.0804153.7499027.3202526.98973913.27972312.9492121.11172220.78120929.28098628.950473
304.50.3724824.407784.0352987.9434917.57100914.41685714.04437522.83681222.4643331.62881331.256331
3050.4190234.7606844.3416618.6074998.18847615.62617115.20714824.68870724.26968434.15615133.737128
305.50.4716975.1454844.6737879.3101028.83840516.90937416.43767726.64042326.16872636.84971736.37802
3060.5283815.56165.03321910.0493189.52093718.27229517.74391428.69296128.1645839.69243939.164058
306.50.5881486.0182165.43006810.81965410.23150619.71284919.12470130.88251930.29437142.7216842.133532
3070.6514766.5094985.85802211.62799210.97651621.22598720.57451133.17250532.52102945.96168345.310207
307.50.7150647.0289856.31392112.48628811.77122422.81525522.10019135.58110234.86603849.37452148.659457
3080.7776297.5684746.79084513.4059312.62830124.48141423.70378538.07764737.30001852.98463652.207007
308.50.8418868.1366677.29478114.38554213.54365626.22172225.37983640.68761839.84573256.7480955.906204
3090.9108148.7339597.82314515.43676514.52595128.03934827.12853443.43279742.52198360.63370359.722889
309.50.9797479.3488158.36906816.55174315.57199629.93052628.95077946.33647645.35672964.70098663.721239
3101.0529859.9935928.94060717.72367716.67069231.89769530.8447149.35513248.30214768.94142667.888441
310.51.12894310.6571059.52816218.95134517.82240233.93116532.80222252.48687951.35793673.30454672.175603
3111.20363811.35354710.14990920.21858619.01494836.03724634.83360855.76286954.55923177.84188876.63825
311.51.27524312.08126510.80602221.51656120.24131838.24850736.97326459.12339757.84815482.57554881.300305
3121.3413212.83383911.49251922.83991621.49859640.54869439.20737462.63997161.29865187.48911986.147799
312.51.4019313.60030712.19837724.19925622.79732642.9256941.5237666.28674964.88481992.62235791.220427
3131.45789714.37843912.92054225.5908624.13296345.37844943.92055270.04007868.58218197.95701796.49912
313.51.50916815.16602913.65686127.03054125.52137347.93026146.42109373.93719772.428029103.471745101.962577
3141.55036715.95624314.40587628.51411826.96375150.57813349.02776677.98335576.432988109.193128107.642761
314.51.58859116.76130915.17271830.03656628.44797553.27802651.68943582.14990680.561315115.083112113.494521
3151.62283917.56185815.93901931.60418529.98134656.00403154.38119286.47387784.851038121.074565119.451726
315.51.6539118.36800316.71409333.19967731.54576758.74179257.08788290.90596789.252057127.150642125.496732
3161.67664519.18905917.51241434.8022833.12563561.54673659.87009195.3737593.697105133.292232131.615587
316.51.688120.02709618.33899636.41161334.72351364.4167162.7286199.9132698.22516139.454796137.766696
3171.68731620.8743319.18701438.03359136.34627567.33232565.645009104.517087102.829771145.680434143.993118
317.51.67785121.72308720.04523639.66465137.986870.26842668.590575109.123967107.446116152.002493150.324642
3181.65958822.57846920.91888141.30207239.64248473.24171.581412113.809483112.149895158.365797156.706209
318.51.62729923.44783421.82053542.95746241.33016376.28264374.655344118.490301116.863002164.873876163.246577
3191.58827924.34486622.75658744.64363343.05535479.37090577.782626123.136889121.54861171.52424169.935961
319.51.5401225.26245623.72233646.39500944.85488982.47606980.935949127.886727126.346607178.311649176.771529
3201.48778426.1720424.68425648.19734546.70956185.58048684.092702132.708429131.220645185.261878183.774094
320.51.43151527.06103825.62952350.03186748.60035288.73911187.307596137.537459136.105944192.141519190.710004
3211.37054727.94313926.57259251.89980650.52925991.96286590.592318142.371398141.000851198.965641197.595094
321.51.30394928.82535527.52140653.79194452.48799595.24096593.937016147.229535145.925586205.77401204.470061
3221.23453429.67959628.44506255.68072754.44619398.55599797.321463152.071789150.837255212.529652211.295118
322.51.16371830.5061129.34239257.55106556.387347101.807071100.643353156.967597155.803879219.002715217.838997
3231.08859231.32145230.2328659.40795558.319363105.029333103.940741161.811408160.722816225.306517224.217925
323.51.01337432.14353931.13016561.25058960.237215108.216203107.202829166.503724165.49035231.494407230.481033
3240.93551632.99872232.06320663.09233862.156822111.295935110.360419171.187025170.251509237.596126236.66061
324.50.85854333.87888533.02034264.93234164.073798114.32607113.467527175.905241175.046698243.747562242.889019
3250.78671734.75698233.97026566.7644565.977733117.288911116.502194180.566628179.779911249.734639248.947922
325.50.72189935.64909834.92719968.60445967.88256120.226812119.504913185.109834184.387935255.653893254.931994
3260.66249336.54975935.88726670.44013569.777642123.16998122.507487189.636505188.974012261.613026260.950533
326.50.60848137.43898636.83050572.23566771.627186126.187625125.579144194.113282193.504801267.782319267.173838
3270.56137838.30242737.74104974.01779973.456421129.204503128.643125198.611198.049622274.018876273.457498
327.50.51763539.14404938.62641475.80801475.290379132.178958131.661323203.068188202.550553280.325295279.80766
3280.47732839.96484539.48751777.5643477.087012135.168428134.6911207.48086207.003532286.816561286.339233
328.50.44031140.77650640.33619579.29414178.85383138.126234137.685923211.943094211.502783293.341866292.901555
3290.40445141.59496341.19051281.01364980.609198141.124212140.719761216.595542216.191091299.951529299.547078
329.50.37181842.40624642.03442882.74571382.373895144.111031143.739213221.309561220.937743306.357598305.98578
3300.34257543.2327242.89014584.49862684.156051147.109101146.766526226.001771225.659196312.608981312.266406
330.50.31980344.09025443.77045186.28931485.969511150.123907149.804104230.832136230.512333318.755678318.435875
3310.30347244.96034744.65687588.06840187.764929153.23406152.930588235.721319235.417847324.806829324.503357
331.50.29338845.8238345.53044289.82131989.527931156.409063156.115675240.59455240.301162330.797695330.504307
3320.28720346.6979646.41075791.66285491.375651159.600202159.312999245.414284245.127081336.841457336.554254
332.50.28444347.59094847.30650593.53937593.254932162.791756162.507313250.12461249.840167343.163714342.879271
3330.286148.47454748.18844795.4301395.14403166.006709165.720609254.822204254.536104349.589072349.302972
333.50.28918949.35862749.06943897.37482997.08564169.251131168.961942259.475905259.186716356.050526355.761337
3340.29426250.23812449.94386299.36093299.06667172.453367172.159105264.062773263.768511362.402779362.108517
334.50.29968751.14680650.847119101.300915101.001228175.646262175.346575268.557047268.25736368.767402368.467715
3350.30588852.11167151.805783103.22362102.917732178.825965178.520077273.210854272.904966375.109536374.803648
335.50.31272653.10045452.787728105.115814104.803088182.021157181.708431277.988825277.676099381.305389380.992663
3360.3196154.07782253.758212106.865303106.545693185.197714184.878104282.788945282.469335387.333443387.013833
336.50.32596955.03729354.711324108.554984108.229015188.357622188.031653287.48792287.161951393.10257392.776601
3370.33213356.0113755.679237110.201862109.869729191.453135191.121002291.977576291.645443398.906062398.573929
337.50.33975256.96450856.624756111.773394111.433642194.609056194.269304296.403765296.064013404.642546404.302794
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4930.0756720.7127890.6371171.8853031.8096312.3799892.3043173.6111213.5354495.1031045.027432
493.50.0793940.7043160.6249221.8816371.8022432.3491442.269753.5844523.5050585.0699524.990558
4940.0802680.6953910.6151231.8746741.7944062.3163652.2360973.5640693.4838015.0228754.942607
494.50.0794750.6863840.6069091.8649881.7855132.2838752.20443.5452343.4657594.9717314.892256
4950.0782450.6793970.6011521.8475991.7693542.252212.1739653.5320383.4537934.9200754.84183
495.50.0762770.6755970.599321.8269181.7506412.2204142.1441373.5146223.4383454.8697044.793427
4960.0723670.6703590.5979921.8042141.7318472.1942532.1218863.488293.4159234.8240224.751655
496.50.0679960.6648340.5968381.7833391.7153432.179732.1117343.4570193.3890234.7825424.714546
4970.0639360.6619280.5979921.7635311.6995952.1750622.1111263.4225833.3586474.7482754.684339
497.50.061170.6610560.5998861.7456321.6844622.1730062.1118363.3826483.3214784.7187464.657576
4980.0598450.6610810.6012361.7291741.6693292.1725412.1126963.3525173.2926724.6855344.625689
498.50.0589760.6615380.6025621.7128991.6539232.1751512.1161753.3301113.2711354.6379284.578952
4990.0584750.6614170.6029421.6947471.6362722.1807392.1222643.3204433.2619684.5768854.51841
499.50.0599340.6632430.6033091.6731121.6131782.1852422.1253083.3256913.2657574.5085234.448589
5000.0630.6680.6051.6481.5852.1852.1223.3383.2754.4374.374

Figure 2: Blank Corrected Lysozyme Standard Fluorescence by Wavelength

Description of image

Then, the areas underneath the fluorescence emission curve in Figure 2 were integrated using the trapezoid method.

    1. The trapezoid method involves calculating the trapezoidal area incrementally and summing all those areas through the formula (B2+B1)/2*(A2-A1) where A1 is the first wavelength (x-axis) data point and B1 is the first emission intensity (y-axis) data point. Note that the last trapezoid area is not included in the summation as the calculation is skewed by the absence of following data that contributes to the calculation. These areas are shown in Table 4 below.

Table 4: Integrated Lysozyme Fluorescence Spectra by Sample Concentration

Concentration (uM) Integrated Emission Intensity
0.9654968.558802
1.939575.247763
3.8616960.03477
7.22526108.92364
15.44535711.23678

Figure 3: Lysozyme Integrated Standard Fluorescence Intensity by Standard Concentration

Description of image

4) Protease (alpha chymotrypsin) samples were prepped for future experiments with the following procedure (x15 tubes)

    1. Mass was recorded 1 eppendorf tube and then balanced tared
    2. roughly 1 mg of alpha chymotrypsin was added and mass recorded
    3. tube was labeled with protease and concentration after future addition of 1mL water
    4. samples placed in Group 4 (JNB) labeled freezer box and stored for later use

Table 5: Alpha Chymotrypsin Prepped Sample Data

Eppendorf Tube Mass (g) Mass of protease (g) Final Molarity in 1mL H2O
1.0150.0013753.515625
1.013480.0013151.171875
1.01790.0010541.015625
1.029540.0013552.734375
1.02280.0010239.84375
1.020480.001350.78125
1.019450.0011143.359375
1.027230.0009938.671875
1.029510.0010541.015625
1.015170.0010842.1875
1.029540.0009436.71875
1.032110.0010440.625
1.01920.0011143.359375
1.027810.0012147.265625

Matt Hartings This is some enthusiasm here!! You really don't need to put the raw data into your lab notebook pages. Just the graphs should be fine. For your A vs concentration graph, please just show your data in points. Keep the fit as a line. Also what is your molar absorptivity in M-1 cm-1?

Data


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