User:Benjamin Friedel/Notebook/CHEM 471/2015/09/29: Difference between revisions
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# Both samples were then incubated in a 37˚C water bath for 1 hour. | # Both samples were then incubated in a 37˚C water bath for 1 hour. | ||
#'Fluorescence Sample and Blank Standards' | |||
1.5mL Eppindorf tubes were created and labeled A-H for both the blank and the lysozyme samples (16 tubes total). Sample A was pure solution of either the blank or lysozyme added solutions created above. A serial dilution of both the blank and lysozyme solutions for samples B-H by diluting the concentrations by one half each time (75mL of HPLC water and 75mL of the previous standard solution). | |||
In separate 1.5 mL Eppindorf tubes, each solution created consisted of 20µM of blank or lysozyme added solution along with 140µM of Assay Buffer and 40µM of Assay Reagent. These new solutions were then individually measured in 1mL fluorescence cuvette on the fluorometer at the following specifications: excitation: 390nm, emission: 400-650nm. | |||
==Data== | ==Data== |
Revision as of 15:34, 7 October 2015
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ObjectiveToday's objective is to monitor the degradation lysozyme by our protease ( alpha-chymotrypsin) over time through measuring fluorescence of lysozyme samples incubated in the with the protease over different time intervals. This experiment is controlled through also monitoring samples containing no lysozyme in the same conditions.
ProcedureDr. Hartings's protocol for the fluorescence experiment can found in his notebook.
1.5mL Eppindorf tubes were created and labeled A-H for both the blank and the lysozyme samples (16 tubes total). Sample A was pure solution of either the blank or lysozyme added solutions created above. A serial dilution of both the blank and lysozyme solutions for samples B-H by diluting the concentrations by one half each time (75mL of HPLC water and 75mL of the previous standard solution). In separate 1.5 mL Eppindorf tubes, each solution created consisted of 20µM of blank or lysozyme added solution along with 140µM of Assay Buffer and 40µM of Assay Reagent. These new solutions were then individually measured in 1mL fluorescence cuvette on the fluorometer at the following specifications: excitation: 390nm, emission: 400-650nm. Data |