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M9195

Sigma-Aldrich

Matrix Metalloproteinase-1 human

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

Synonym(s):
Collagenase-1, Interstitial Collagenase, MMP-1
Enzyme Commission number:
MDL number:
NACRES:
NA.32

recombinant

expressed in NSO cells

Quality Level

assay

>95% (SDS-PAGE)

form

buffered aqueous solution

enzyme activity

≥400 pmol/min-μg

mol wt

apparent mol wt 52-55 kDa

UniProt accession no.

shipped in

dry ice

storage temp.

−70°C

Gene Information

human ... MMP1(4312)

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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

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US), Eyeshields, Gloves

Certificate of Analysis

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Certificate of Origin

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Product Information Sheet

More Documents

SDS

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