MMP-9 Protein, Mouse (HEK293)

The MMP-9 protein is a matrix metalloproteinase that is critical for local extracellular matrix proteolysis and leukocyte migration.It is suggested that it may be involved in bone osteoclastic resorption.MMP-9 Protein, Mouse (HEK293) is the recombinant mouse-derived MMP-9 protein, expressed by HEK293 , with tag free.

MMP-9 Protein, a matrix metalloproteinase, plays a crucial role in local proteolysis of the extracellular matrix and facilitates leukocyte migration. It could be involved in bone osteoclastic resorption and cleaves KiSS1 at a specific Gly-|-Leu bond. Additionally, MMP-9 cleaves NINJ1 to generate the Secreted ninjurin-1 form and processes type IV and type V collagen into large C-terminal three-quarter fragments and shorter N-terminal one-quarter fragments. While degrading fibronectin, MMP-9 does not impact laminin or Pz-peptide, showcasing its selectivity in substrate cleavage.

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH2. The specific activity is >1,500 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated by APMA (HY-148905) for an activated form.)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v) , pH 7.5 (TCNB)
MMP-9 Protein, Mouse (HEK293) (HY-P73807)
p-aminophenylmercuric acetate (APMA, HY-148905), diluted with DMSO to 100 mM for storage
Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (HY-131498)
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard curve: Dilute MCA-Pro-Leu-OH (standard) to 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM with assay buffer, and add 100 μL of each into black enzyme-labeled wells. The excitation and emission wavelengths were 320 nm and 405 nm, respectively, and read in kinetic mode for 5 min. The measured RFU was used as the horizontal coordinate, and the amount of standard substance was used as the horizontal coordinate to make the standard curve and obtain the standard curve formula.
2. Proteins were solubilized to 1 mg/mL with ddH₂O and subsequently diluted to 100 μg/mL in assay buffer.
3. Mouse MMP-9 was activated by addition of APMA to a final concentration of 1 mM APMA .
4. Incubate at 37°C for 2 hours.
5. Dilute the activated MMP-9 to 0.4 μg/mL in the assay buffer.
6. The substrate was diluted to 20 μM in the assay buffer.
7. Experimental group: 50 μL of 0.4 μg/mL Mouse MMP-9 was added to the plate and the reaction was started by adding 50 μL of 20 μM substrate.
Control: 50 μL of assay buffer and 50 μL of 20 μM substrate.
8. Readings were taken in kinetic mode for 5 min at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
9. Calculate specific activity:

Mouse

HEK293

Tag Free

P41245 (P21-P730)

17395  [NCBI]

Partial

PYQRQPTFVVFPKDLKTSNLTDTQLAEAYLYRYGYTRAAQMMGEKQSLRPALLMLQKQLSLPQTGELDSQTLKAIRTPRCGVPDVGRFQTFKGLKWDHHNITYWIQNYSEDLPRDMIDDAFARAFAVWGEVAPLTFTRVYGPEADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGAGVQGDAHFDDDELWSLGKGVVIPTYYGNSNGAPCHFPFTFEGRSYSACTTDGRNDGTPWCSTTADYDKDGKFGFCPSERLYTEHGNGEGKPCVFPFIFEGRSYSACTTKGRSDGYRWCATTANYDQDKLYGFCPTRVDATVVGGNSAGELCVFPFVFLGKQYSSCTSDGRRDGRLWCATTSNFDTDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPLYSYLEGFPLNKDDIDGIQYLYGRGSKPDPRPPATTTTEPQPTAPPTMCPTIPPTAYPTVGPTVGPTGAPSPGPTSSPSPGPTGAPSPGPTAAPTAGSSEASTESLSPADNPCNVDVFDAIAEIQGALHFFKDGWYWKFLNHRGSPLQGPFLTARTWPALPATLDSAFEDPQTKRVFFFSGRQMWVYTGKTVLGPRSLDKLGLGPEVTHVSGLLPRRPGKALLFSKGRVWRFDLKSQKVDPQSVIRVDKEFSGVPWNSHDIFQYQDKAYFCHGKFFWRVSFQNEVNKVDPEVNQVDDVGYVTYDLLQCP

Approximately 80-105 kDa

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder

Lyophilized from a 0.2 μm filtered solution of PBS, pH 7.4 or 20 mM PB, 150 mM NaCl, pH 7.4 or PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.