User:Jamie Nunziata/Notebook/Protease Research/2015/09/09

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Objective

The objective of today's lab work included making stock solutions of lysozyme of varying concentrations and analyze the solutions using the data collected from UV-Vis and Fluorescence Spectrometers. Finally, we prepped 15 1mL alpha chymotrypsin samples and stored them in the freezer for future use. Details on today's protocols can be found in Dr. Harting's notebook here.


Procedure

The solutions used in this experiment were of the concentrations 15.445µM, 7.225µM, 3.86µM, 1.93µM, and 0.965µM. They were made by first adding 4.42mg of lysozyme to 10mL of HPLC water, making a 30.89µM solution. A serial dilution was then performed by taking 5mL of the previous concentrated solution and adding another 5mL of HPLC water until we had 5 solutions under the concentration of 20mL. These solutions were labeled and store in 15mL Falcon tubes. Prior to analyzing these samples using a UV Vis and Fluorescence Spectrometer, the cuvette used first had to be tested with just HPLC water to blank the data later on. The measurements gathered from these tests are shown below in the Data section

To prep the 15 1mL samples of alpha-chymotrypsin, our group's protease of study, the Eppindorf tubes used were first weighed and stored in a speadsheet to find an average mass and standard deviation. Then, about 1mg of dry alpha-chymotrypsin was added to the tubes with 1mL of HPLC water. The samples were then stored in the freezer.


Data

Figure 1: UV Vis calibration curve at 280nm

Nunziata CCurve 20150909.png


Figure 2: UV Vis data, corrected for the blank

Nunziata UVVis 20150909.png


Figure 3: Fluorescence data, corrected for the blank

Nunziata Fluorescence 20150909.png


Figure 4: Integrated Fluorescence data

Nunziata FluorescenceIntegration 20150909.png


Matt Hartings List the molar absorptivity of lysozyme in M-1 cm-1. Also, change your integrated fluorescence vs concentration plot so that it shows points instead of a line.