- To make standardized curves for determining peptide concentration using fluorescence analysis.
- To run blanks for UV/Vis experiments we did yesterday.
We used the Pierce quantitative fluorometric peptide assay. (product link here)
To perform a protease digest of lysozyme
- To make a stock lysozyme solution we weighed out 5.14 mg of lysozyme and placed it into a 5 mL volumetric flask and pipetted to the line with phosphate buffer.
- We retrieved one of our pre-measured protease (trypsin) and added 1 mL phosphate buffer to create a 53.22 µM protease (trypsin) stock.
- We combined 0.0188 mL of the 53.22 µM protease stock with 0.9812 mL stock lysozyme solution to create a 1 µM protease reaction sample solution.
- We combined 0.0188 mL of protease stock with .9812 mL of phosphate buffer to create a blank sample (no lysozyme).
- We placed both tubes (reaction sample and blank sample) to a 37C water bath for 1hr
- To make standards for analysis
- Standard A: 150uL of reaction sample we
- 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
- To make blanks for analysis we
- Blank A: 150uL of blank sample
- 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
- To create samples for measurement we
- For each of the standards and blanks described above mix the following
- 20uL of sample
- 140uL of Assay Buffer
- 40uL of Assay Reagent
- We took measurements at
- Excitation at 390 nm
- Emission from 400 to 650 nm
- We followed the procedure here except we did not use any gold fibers.