M9195
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
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:
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
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
WGK
WGK 2
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Personal Protective Equipment
dust mask type N95 (US), Eyeshields, Gloves
Regulatory Information
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Interstitial collagenase.
Cawston, T.E. et al.
Handbook of Proteolytic Enzymes, 472-480 (2004)
N S Templeton et al.
Cancer research, 50(17), 5431-5437 (1990-09-01)
A full-length complementary DNA (cDNA) for interstitial collagenase was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The tumor interstitial collagenase cDNA was sequenced and compared to the published sequences for human fibroblast collagenase. The sequence
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