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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

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About This Item

Enzyme Commission number:
MDL number:
UNSPSC Code:
12352202
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

10 - 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

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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