MMP-9(20-469) His Tag Protein, Cynomolgus

Matrix metalloproteinase-9 (MMP-9, also known as 92 kDa gelatinase B) is a structurally complex key protease capable of degrading extracellular matrix components such as type IV collagen and gelatin. Secreted by various cells including neutrophils and macrophages, it plays vital roles in both normal tissue remodeling and pathological inflammatory processes. The activity of this enzyme requires precise regulation, and its overexpression can disrupt tissue homeostasis, thereby actively contributing to the pathogenesis of various diseases such as rheumatoid arthritis, multiple sclerosis, and tumor invasion. Consequently, it is regarded as a highly potential and important therapeutic target.

Experimental Method

Assay Principle: Detects the ability ofMMP-9protein to cleaveMca-PLGL-Dpa-AR-NH2.

Materials

1. Assay Buffer:50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)

2. MMP-9(20-469) His Tag Protein, Cynomolgus

3. p-aminophenylmercuric acetate (APMA) (Aladdin, Catalog # P649286)

4. Substrate:MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (R&D, ES001)

5. Standard:MCA-Pro-Leu-OH (Shyuanye, T77046)

6. 96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)

7. Plate Reader (PerkinElmer, Excitation Wavelength/Emission Wavelength=320 nm/405 nm)

Procedure

1. In the assay buffer containing1mM APMA, dilute theMMP-9protein to100 μg/mL, and incubate at37°Cfor4hours to activate theMMP-9protein.

2. Dilute the activatedMMP-9protein with assay buffer to0.8, 0.4, 0.2 μg/mL.

3. Dilute the substrate in buffer to20 μM.

4. Add50 μLof the dilutedMMP-9protein (from Step 2), then add50 μL 20 μMsubstrate to initiate the reaction. Include wells containing only substrate and assay buffer as blank controls.

5. At320 nmexcitation wavelength and405 nmemission wavelength, read the fluorescence signal in kinetic mode (60seconds/cycle,20cycles).

6. Calculate Specific Activity:

Standard Curve

1. Dilute the standard with assay buffer to10 μM, and perform serial dilutions.

2. Add100 μLof the serially diluted standards and blank (assay buffer) to the corresponding wells of the96-well plate. The concentration range for the standard curve is per well1000,500,250,125,62.5,31.25,15.625 pmol.

3. At excitation wavelength320 nmand emission wavelength405 nm, read the fluorescence signal in endpoint mode.

4. Plot theMCA-Pro-Leu-OHpmolamount on the y-axis(y), and the relative fluorescence units (RFU) after blank subtraction on the x-axis (x), then perform linear regression analysis.