Objective
Today'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.
Procedure
Dr. Hartings's protocol for the fluorescence experiment can found in his notebook.
- First, a stock solution of lysozyme was made in 50µM phosphate buffer
- 5.2mg of dry lysozyme was dissolved 5mL of phosphate buffer using a 5mL volumetric flask to realize a final concentration of 1.04mg/mL (target=1.00mg/mL)
- Second, a stock solution of our protease (alpha-chymotrypsin was made)
- 1mL of HPLC grade water was added to a previously measured sample for a final stock concentration of 39.84uM
- Then, the a stock sample of an alpha-chymotrypsin and lysozyme reaction mix was made for incubation and fluorescence testing
- This sample contained 1µM protease and 974.9ul of the 1.04mg/mL lysozyme (for a total volume of 1ml)
- Through M1V1=M2V2 the volume of protease to be added from the stock solution was determined to be 25.1ul
- Volume of lysozyme to bring the final sample volume to 1mL was calculated by subtracting the volume of protease from 1ml. Thus 974.9ul of lysozyme stock was added to the 1.5mL eppendorf tube for the reaction mix.
- A made a blank solution of an alphachymotrypsin and phosphate buffer was then prepared to account for self degradation by the protease as well as fluorescence from the protease alone.
- Following the sample procedure, 25.1uL of protease was added to 974.9uL of phosphate buffer for a total protease concentration of 1uM.
- Both samples were then incubated in a 37˚C water bath for 1 hour.
Data
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