ADAM8 Protein, Rhesus Macaque (HEK293, His)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
ADAM8 Protein, Rhesus Macaque (HEK293, His) (HY-P772930)
Bacterial Thermolysin
Phosphoramidon: Dissolve in methanol to a concentration of 20 mM Substrate: Mca-PLAQAV-Dpa-RSSSR-NH2 (HY-P3722A)
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard curve preparation: Dilute MCA-Pro-Leu-OH (standard) in assay buffer to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, 0.39, 0.19, 0 pmoL. Add 100 μL of each standard concentration to a 96-well plate. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively. Plot the standard concentration on the x-axis and the measured RFU on the y-axis to generate a standard curve and obtain the standard curve equation.
2. Dilute the test protein to 400 μg/mL in assay buffer.
3. Mix equal volumes of 1.5 μg/mL Thermolysin and diluted test protein.
4. Incubate at 37°C for 30 minutes.
5. Add Phosphoramidon to a final concentration of 0.05 mM to stop the reaction.
6. Incubate at room temperature for 15 minutes.
7. Dilute the activated test protein to 40 ng/μL in assay buffer.
8. Dilute the substrate to 40 μM in assay buffer.
9. Load 50 μL of 40 ng/μL test protein into a black plate and add 50 μL of 40 μM substrate. For the blank, add 50 μL of assay buffer and 50 μL of 40 μM substrate.
10. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
11. Calculate the specific activity: