MMP-9 Protein, Human (HEK293, His, solution)

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, Human (HEK293, His, solution) is the recombinant human-derived MMP-9 protein, expressed by HEK293 , with C-6*His labeled tag.

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.

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, Human (HEK293, His, solution) (HY-P70145)
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 μMol/L 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 material was used as the horizontal coordinate to make the standard curve and obtain the standard curve formula.
2. Dilute the Human MMP-9 to 100 μg/mL in assay buffer.
3. Activate Human MMP-9 by adding APMA so that the final concentration of APMA was 1 mM.
4. Incubate at 37°C for 24 hours.
5. Dilute the activated Human MMP-9 to 0.08 μg/mL in assay buffer.
6. The substrate was diluted to 20 μM in assay buffer.
7. Experimental group: 50 μL of 0.08 μg/mL Human 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 the specific activity:

Human

HEK293

C-6*His

AAH06093.1 (A19-D707)

4318  [NCBI]

Partial

AAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGESKSLGPALLLLQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTRDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPEPRPPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEEPLSKKLFFFSGRQVWVYTGASVLGPRRLDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYWRVSSRSELNQVDQVGYVTYDILQCPED

Approximately 90.0 kDa

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

<1 EU/μg, determined by LAL method.