Recombinant Human MMP-9 His-tag Avi-tag Protein, CF

Recombinant Human MMP-9 His-tag Avi-tag (Catalog # AVI911) is measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH2 (ES001).

Biotinylated Recombinant Human MMP-9 His-tag Avi-tag Protein (Catalog # AVI911) binds Human/Primate MMP-9 Antibody (MAB936R) with an ED50 of 1.25-15.0 ng/mL.

2 μg/lane of Biotinylated Recombinant Human MMP‑9 His-tag Avi-tag Protein (Catalog # AVI911) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 90-100 kDa under reducing conditions.

• Reactivity: Hu
• Applications: Binding Activity, Enzyme Activity
• Format: Carrier-Free

Recombinant Human MMP-9 His-tag Avi-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH₂ (Catalog # ES001). The specific activity is >1500 pmol/min/μg, as measured under the described conditions. Measured by its binding ability in a functional ELISA. Biotinylated Recombinant Human MMP-9 His-tag Avi-tag (Catalog # AVI911) binds Human/Primate MMP-9 Antibody (Catalog # MAB936R) with an ED₅₀ of 1.25-15.0 ng/mL.
• Source: Chinese Hamster Ovary cell line, CHO-derived human MMP-9 protein
  

  Human MMP-9 (Ala20-Asp707) Accession # P14780.1	6-His tag	GG	Avi-tag
  N-terminus			C-terminus

• N-terminal Sequence: Ala20
• Structure / Form: Proform
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Binding Activity2
  • Enzyme Activity
• Theoretical MW: 79.0 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 90-100 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, CaCl₂, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
  • Recombinant Human MMP-9 His-tag Avi-tag (rhMMP-9) (Catalog # AVI911)
  • p-aminophenylmercuric acetate (APMA), 100 mM stock in DMSO
  • Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES001)
  • Black 96-well Plate
  • Plate Reader with Fluorescence Read Capability
  1. Dilute rhMMP-9 to 100 µg/mL in Assay Buffer.
  2. Activate rhMMP-9 by adding APMA to a final concentration of 1 mM.
  3. Incubate at 37 °C for 24 hours.
  4. Dilute activated rhMMP-9 to 0.4 µg/mL in Assay Buffer.
  5. Dilute Substrate to 40 µM in Assay Buffer.
  6. Load 50 µL of 0.4 µg/mL rhMMP-9 into a plate and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 40 µM Substrate.
  7. Read at excitation 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
  Per Well:
  rhMMP-9: 0.02 µg
  Substrate: 20 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human MMP-9 His-tag Avi-tag Protein, CF

• 92 kDa gelatinase
• 92 kDa type IV collagenase
• CLG4B
• EC 3.4.24
• EC 3.4.24.35
• Gelatinase B
• GELB
• macrophage gelatinase
• MANDP2
• matrix metallopeptidase 9
• matrix metalloproteinase 9
• matrix metalloproteinase-9
• MMP9
• MMP-9
• type V collagenase

Background

Recombinant human Active Matrix Metalloproteinase 9 (MMP-9), also known as gelatinase B, is a member of the MMP family of zinc and calcium-dependent endopeptidases. MMP-9 protein is synthesized as a pre-proenzyme in specific cells such as neutrophils, macrophages, fibroblasts and endothelial cells (1). Expressed MMP-9 contains a signal peptide to transport it to the extracellular matrix (ECM), a hinge region, a propeptide region, a catalytic domain, and a hemopexin-like domain that is important for substrate recognition (2,3).  In addition to having three fibronectin type II domains that contribute to substrate binding and an active site, the catalytic domain of MMP-9 also contains a zinc-binding region that interacts with a cysteine in the propeptide to maintain latency.  Consequently, removal of the propeptide through cleavage by proteases in the ECM, such as MMP3, activates the protein (2, 3). MMP-9 has specificity for targets containing an established preferred consensus sequence (3-5) and has a broad range of substrates within the ECM including gelatin, collagen, elastin that contributes to its role in ECM remodeling and extracellular domain cell surface protein release from the plasma membrane (3). Due to its activity in the ECM, MMP-9 plays a role in many biological processes and can serve as a biomarker in tumor invasion and mestastasis (3,6) of many cancers including colon, ovarian, breast, osteosarcoma, and lung cancers (7-11) making it a therapeutic target (6, 8, 12). MMP-9 modeling of the ECM also leads to a pivotal role in other inflammation- and autoimmune-related diseases (3, 13, 14). Our Avi-tag Biotinylated human MMP-9 His-tag features biotinylation at a single site contained within the Avi-tag, a unique 15 amino acid peptide. Protein orientation will be uniform when bound to streptavidin-coated surface due to the precise control of biotinylation and the rest of the protein is unchanged so there is no interference in the protein's bioactivity.

1. Vandooren, J. et al. (2013) Crit. Rev. Biochem. Mol. Biol.  48: 222.
2. Roeb, E. et al. (2002) J. Biol. Chem. 277: 50326.
3. Huang, H. (2018) Sensors. 18:3249.
4. Kridel, S.J. et al. (2001) J. Biol. Chem. 276: 20572.
5. Prudova, A. et al. (2010) Mol. Cell Proteom. 9:894.
6. Kalali, D. (2023) Glob. Med. Genet. 10:48.
7. Hu, X. et al. (2012) Arch. Glynecol. Obstet. 286:1537.
8. Wang, J. et al. (2014) Clin. Chim. Acta. 433:225.
9. Blanco-Prieto, S. et al. (2017) BMC Cancer 17:823.
10. Liang, S. and L. Chang. (2018) Biomark. Med. 12:393.
11. Malik, S. et al. (2024) Sci. Rep. 14:15117.
12. Roy, R. et al. (2009) J. Clin. Oncol. 27:5287.
13. Ram, M. et al. (2006) J. Clin. Immunol. 26:299.
14. Kim, I.S. et al. (2023) Curr. Med. Chem. 30:2075.

FAQs for MMP-9 (AVI911). (Showing 1 - 1 of 1 FAQ).

1.  I’m looking for a pair of antibodies to MMP-9 that can be used in a sandwich assay. Do you carry any? 
   • We have 10 primary antibodies for MMP-9 that have been tested in ELISA, seen here.It looks like 2 have been tested for capture (please note the tested species for each of these), seen here.6 have been tested for detection, seen here.