MMP-9 Protein, Mouse (HEK293)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v) , pH 7.5 (TCNB)
MMP-9 Protein, Mouse (HEK293) (HY-P73807)
p-aminophenylmercuric acetate (APMA, HY-148905), diluted with DMSO to 100 mM for storage
Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (HY-131498)
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard curve: Dilute MCA-Pro-Leu-OH (standard) to 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM with assay buffer, and add 100 μL of each into black enzyme-labeled wells. The excitation and emission wavelengths were 320 nm and 405 nm, respectively, and read in kinetic mode for 5 min. The measured RFU was used as the horizontal coordinate, and the amount of standard substance was used as the horizontal coordinate to make the standard curve and obtain the standard curve formula.
2. Proteins were solubilized to 1 mg/mL with ddH₂O and subsequently diluted to 100 μg/mL in assay buffer.
3. Mouse MMP-9 was activated by addition of APMA to a final concentration of 1 mM APMA .
4. Incubate at 37°C for 2 hours.
5. Dilute the activated MMP-9 to 0.4 μg/mL in the assay buffer.
6. The substrate was diluted to 20 μM in the assay buffer.
7. Experimental group: 50 μL of 0.4 μg/mL Mouse MMP-9 was added to the plate and the reaction was started by adding 50 μL of 20 μM substrate.
Control: 50 μL of assay buffer and 50 μL of 20 μM substrate.
8. Readings were taken in kinetic mode for 5 min at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
9. Calculate specific activity: