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M9195 Sigma-Aldrich

Matrix Metalloproteinase-1 human

recombinant, expressed in NSO cells, >95% (SDS-PAGE), buffered aqueous solution

Synonym: Collagenase-1, Interstitial Collagenase, MMP-1

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Description

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

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
2

Documents

Certificate of Analysis

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Certificate of Origin

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Milli-Q® Water Purification Solutions
Protocols & Articles
Peer-Reviewed Papers
15

References

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