Objectives
- To determine the molar absorptivity of lysozyme using UV-Vis. This data is useful as we move on to protein concentration with the Bradford assay in the future.
- To determine how protein concentration affects fluorescence intensity.
- Prepare a protease sample.
Procedure
Protocols
- Lysozyme samples for spectral analysis
- Lysozyme has a molecular weight of 14307 g/mol
- We prepared a 51.2uM lysozyme stock soluton by weighing out 7.32 mg of lysozyme and transfered it to a 10 mL volumetric flask where we pipetted to the mark with deionized water. We transfered our stock solution to a falcon tube.
- We made 5 lysozyme samples for analyzing with UV Vis and fluorescence spectroscopies between a working range on 0 and 15 uM.
- (volume of stock or previous sample)(Conc of used sample
0/(10 mL deionized water)=new sample conc.
- (2.93 mL)(51.2 uM)/10 mL= 15 uM
- (5 mL)(15 uM)/10 mL= 7.5uM
- (7 mL)(7.5 uM)/10 mL=5.25 uM
- (5 mL)(5.25 uM)/10 mL= 2.625 uM
- (3 mL)(2.625uM)/10 mL= 0.7875 uM
- UV Vis data
- We measured a water blank
- We ran our 5 samples within the working range, using the UV Vis and Fluorescence.
- Correct the spectra for solvent and baseline
- Fluorescence data
- Measure a water blank
- Measure the spectrum of each of your samples (except for your 50 uM sample)
- Using Excel, calculate the area underneath the curve (i.e. integrate) for each of your spectra.
- Prepping protease samples
- Mass of 1 eppendorf tube: 1.03215 g
- (Add roughly 1 mg of Trypsin) Mass of Trypsin: 0.00122 g
- Label the tube with the name of the protease and what the concentration will be after you add 1 mL of water.
- mass of trypsin/23300(MW of Trypsin)/.001 L =__ M
- Place in your freezer box
Data
Results
Lysozyme Molar Absorbtivity Coefficient: 37700 M-1 cm-1
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