Biochem/physiol Actions
MMP-1 initiates the breakdown of interstitial collagens.
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.
Analysis Note
The biological activity is measured by its ability to cleave a fluorogenic peptide substrate.
Other Notes
Activity Assay Protocol
Materials:
- 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 equivalent
Assay:
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 Vmax* (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 μg
Substrate: 10 μM
Legal Information
Brij is a registered trademark of Croda International PLC
Shipping Information:
Dry Ice Surcharge & Ice Pack Shipments: $40
More Information: https://cenmed.com/shipping-returns
- UPC:
- 51203100
- Condition:
- New
- Availability:
- 3-5 Days
- Weight:
- 1.00 Ounces
- HazmatClass:
- No
- MPN:
- M9195-10UG
- Temperature Control Device:
- Yes
