User:Jamie Nunziata/Notebook/Protease Research/2015/09/09
|Project name||Main project page|
Previous entry Next entry
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.
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.
Figure 1: UV Vis calibration curve at 280nm
Figure 2: UV Vis data, corrected for the blank
Figure 3: Fluorescence data, corrected for the blank
Figure 4: Integrated Fluorescence data