Analysis Note

The biological activity is measured by its ability to cleave a fluorogenic peptide substrate.

Biochem/physiol Actions

MMP-1 initiates the breakdown of interstitial collagens.

Legal Information

Brij is a registered trademark of Croda International PLC

Other Notes

Activity Assay ProtocolMaterials:•Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)•Recombinant Human MMP-1 (rhMMP-1)(Cat. No. M9195)•p-aminophenylmercuric acetate (APMA), (Cat. No. A9563), 100 mM stock in DMSO•Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 •F16 Black Maxisorp Plate•Fluorescent Plate Reader or equivalentAssay:1. Dilute rhMMP-1 to 50 μg/mL in Assay Buffer.2. Activate 50 μg/mL rhMMP-1 by adding APMA to a final concentration of 1 mM.3. Incubate at 37 °C for 2 hours.4. Dilute activated rhMMP-1 to 1 ng/μL in Assay Buffer.5. Dilute Substrate to 20 μM in Assay Buffer.6. Load into a black well plate 50 μL of 1 ng/μL rhMMP-1 and start the reaction by adding 50 μL of 20 μM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer, 50 μL Substrate, and no rhMMP-1.7. Read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.8. Calculate specific activity: Specific Activity (pmol/min/μg) =Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU) / amount of enzyme (μg)*Adjusted for Substrate Blank**Derived using calibration standard MCA-Pro-Leu-OH.Final Assay Conditions:Per Well:rhMMP-1: 0.050 μgSubstrate: 10 μM

Physical form

Supplied as a 0.2 µm filtered solution of 25 mM MES, 10 mM calcium chloride, 150 mM sodium chloride, 0.05% Brij^® L23, pH 5.5.