First, we'll make a standard curve for the fluorescence of our proteinase, alpha-chymotrypsin. Then, we'll measure the fluorescence of samples of protease + lysozyme and use our standard curve in order to determine how the proteinase degrades the lysozyme.
Dr. Hartings's protocol for today is in his lab notebook.
Today, my group was doing the fluorescence analysis of protein degradation. Our protocol was as follows:
- We made a stock solution of lysozyme in 50µM phosphate buffer
- The target concentration for the stock solution was about 1mg/mL
- We dissolved 5.2mg of dry lysozyme in 5mL of phosphate buffer using a 5mL volumetric flask
- Thus, the actual concentration of our stock was 1.04mg/mL
- We made our protease stock:
- We added 1mL of HPLC water to one of our samples of protease that we had measured out in a previous lab
- The final concentration of our protease (alpha-chymotrypsin) was 39.84µM.
- Next, we made our first sample of alpha-chymotrypsin and lysozyme for use in the fluorescence spectroscopy. (This solution was also the first solution in our serial dilution to make the rest of our samples, as will be shown in step5).
- The target concentration and volume for this sample were 1µM and 1mL, respectively
- First, we determined what volume of alpha-chymotrypsin we would need by doing the following calculation:
M1=concentration of alpha-chymotrypsin=39.84µM
V1=volume of alpha-chymotrypsin needed
M2=concentration of final solution=1µM
V2=volume of final solution=1mL
- Next, we determined what volume of lysozyme to add to the alpha-chymotrypsin to bring the final volume up to 1mL:
Volume of total solution-Volume of alpha-Chymotrypsin=Volume of Lysozyme
- We added both of these components to a 1.5mL Eppindorf tube to make the final solution
- Next, we made a blank solution of our proteinase without lysozyme:
- We wanted the concentration of alpha-chymotrypsin in this sample to be the same as the sample from step 2, and we wanted the final volume of this sample to be 1mL
- Thus, we just needed to add the same volume of alpha-chymotrypsin stock as we did in step 2, which was 25.1µL.
- We brought the total volume of the sample up to 1mL by adding 1000µL-25.1µL=974.6µL of phosphate buffer
- We then incubated both samples in a 37 degree C water bath for 1 hour
- After that, we began making our standards. Our standard samples were made as follows:
- Standard A: 150uL of the reaction sample from step 3
- Standard B: 75uL of Standard A and 75uL of water
- Standard C: 75uL of Standard B and 75uL of water
- Standard D: 75uL of Standard C and 75uL of water
- Standard E: 75uL of Standard D and 75uL of water
- Standard F: 75uL of Standard E and 75uL of water
- Standard G: 75uL of Standard F and 75uL of water
- Standard H: 75uL of Standard G and 75uL of water
- We then made our blanks as follows:
- Blank A: 150uL of the reaction sample from step 4
- Blank B: 75uL of Blank A and 75uL of water
- Blank C: 75uL of Blank B and 75uL of water
- Blank D: 75uL of Blank C and 75uL of water
- Blank E: 75uL of Blank D and 75uL of water
- Blank F: 75uL of Blank E and 75uL of water
- Blank G: 75uL of Blank F and 75uL of water
- Blank H: 75uL of Blank G and 75uL of water
- In separate 1.5mL Eppindorf tubes, we then combined the following (for each standard) just before we took its fluorescence measurements:
- 20uL of a standard
- 140uL of Assay Buffer
- 40uL of Assay Reagent
- We pipetted this solution into the 1mL fluorescence cuvette. We used the same cuvette for each standard; we washed the cuvette out with water, SDS, HCL, and water between each measurement.
- We then measured the fluorescence. Excitation was at 390nm and emission was from 400 to 650nm.
- We did not have enough time to measure our blanks, so we put all of our reagents in the fridge until tomorrow.
See tomorrow's lab entry, as we did not have enough data today to analyze.