Recombinant Human MMP-8

Recombinant Human MMP-8 is widely used in research applications due to its well-characterized role in extracellular matrix (ECM) degradation, its involvement in inflammatory processes, and its utility as a model enzyme for studying collagenase activity and related pathologies.

Key scientific reasons to use recombinant human MMP-8 include:

• ECM Degradation and Collagenase Activity
  MMP-8 is a neutrophil-derived collagenase that efficiently cleaves types I, II, and III collagen, as well as other ECM proteins such as gelatin, fibronectin, proteoglycans, and aggrecan. This makes it a critical tool for studying tissue remodeling, wound healing, and diseases characterized by excessive matrix breakdown (e.g., arthritis, fibrosis, cancer metastasis).

• Inflammation and Disease Modeling
  MMP-8 is released during acute inflammation and is implicated in the pathogenesis of rheumatoid arthritis, cardiovascular disease, sepsis, and chronic wounds. Recombinant MMP-8 allows for controlled in vitro studies of its enzymatic activity, substrate specificity, and effects on cell signaling, enabling mechanistic insights into these conditions.

• Assay Standardization and Controls
  Recombinant MMP-8 is frequently used as a positive control or reference standard in biochemical assays such as zymography, collagenase assays, and enzyme kinetics studies. Its defined activity and purity facilitate reproducible quantification and inhibitor screening.

• Cellular and Molecular Mechanism Studies
  Recombinant MMP-8 can be applied to cell cultures to induce ECM degradation, study pro-inflammatory responses, and investigate downstream effects such as angiogenesis or immune cell recruitment. It is also used in animal models to assess its role in tissue remodeling and behavioral changes.

• Therapeutic Target Validation
  MMP-8 is a potential therapeutic target in diseases where ECM turnover is dysregulated. Recombinant protein enables preclinical testing of inhibitors and exploration of its biological functions.

• Technical Advantages
  Recombinant MMP-8 is highly stable, retains enzymatic activity after storage and freeze-thaw cycles, and is easy to dissolve, supporting consistent and replicable experimental results.

Typical applications include:

• Bioassays for ECM degradation and cell migration
• Zymography and collagenase activity assays
• Enzyme kinetics and inhibitor screening
• In vitro modeling of inflammation and tissue remodeling
• ELISA detection and Western blot controls

In summary, recombinant human MMP-8 is a versatile and reliable tool for investigating ECM biology, inflammatory mechanisms, and for developing and validating therapeutic strategies targeting matrix metalloproteinases.

Yes, recombinant human MMP-8 can be used as a standard for quantification and calibration in ELISA assays, though there are important considerations regarding its application and limitations.

Use as ELISA Standards

Recombinant human MMP-8 is widely employed as the standard material in commercial ELISA kits designed for MMP-8 quantification. These kits are typically produced using NS0-expressed recombinant human MMP-8 in conjunction with antibodies raised against the recombinant protein. The recombinant protein demonstrates dose-response curves that are parallel to natural human MMP-8, indicating that it can reliably determine relative mass values for endogenous MMP-8.

Detection Range and Sensitivity

When using recombinant MMP-8 as a standard, you can expect detection ranges typically spanning from approximately 4.6 pg/mL to 6000 pg/mL, depending on the specific assay formulation. Sensitivity values generally range from 4.6 to 19.5 pg/mL, allowing for detection of both high and low concentrations of the target protein.

Important Limitations

Bioassay Applications: Recombinant MMP-8 standards designated for ELISA use are not recommended for bioassay applications, as they have not been validated for functional activity testing. This distinction is critical if your research requires assessment of enzymatic activity rather than simple protein quantification.

Sample Compatibility: The recombinant standard performs reliably across multiple sample matrices including serum, plasma, cell culture supernatants, saliva, and tissue lysates. However, you should verify that your specific sample type has been validated with the recombinant standard you intend to use.

Practical Considerations

Recombinant MMP-8 standards demonstrate good stability, withstanding room temperature exposure for up to one week and surviving four freeze-thaw cycles without significant loss of activity. This stability makes them practical for routine laboratory use. Endotoxin levels are typically maintained below 1.0 EU per microgram of protein, which is important for maintaining assay specificity.

For optimal results, ensure that your recombinant standard is properly characterized for concentration and purity, and that you follow the manufacturer's recommendations regarding storage conditions and dilution protocols specific to your assay format.

Recombinant Human MMP-8 (Matrix Metalloproteinase-8) has been validated for several key applications in published research, as demonstrated by its use in peer-reviewed studies and technical product reviews. The main applications include:

1. Enzymatic Activity Assays

   • MMP-8 is frequently used in in vitro enzymatic activity assays to study its collagenolytic and proteolytic functions, including quantification of activity using methods such as collagen zymography and activity/RFU assays.
   • Example: Improved collagen zymography for evaluating MMP-8 activity (Ref 8), and activity assays in periodontal disease studies (Ref 6).

2. In Vitro Bioactivity in Cell Culture

   • Recombinant MMP-8 is used to induce biological responses in cultured human cells, such as pro-inflammatory responses and changes in extracellular matrix (ECM) remodeling.
   • Example: Induction of pro-inflammatory and pro-angiogenic responses in ARPE-19 human cells (Ref 1, Ref 11).

3. Animal Studies (In Vivo Applications)

   • MMP-8 has been administered in vivo (e.g., retro-orbital injection in mice) to study its effects on tissue remodeling, neurophysiology, and behavior, particularly in models of stress, inflammation, and disease.
   • Example: Injection of biotinylated recombinant MMP-8 in mice to study brain parenchyma infiltration and behavioral outcomes (Ref 2, Ref 4).

4. Extracellular Matrix (ECM) Remodeling and Degradation Studies

   • MMP-8 is used to investigate ECM breakdown, including degradation of aggrecan and collagen, relevant to diseases such as arthritis, periodontitis, and atherosclerosis.
   • Example: Studies on cartilage degradation, atherosclerotic lesion formation, and periodontal tissue remodeling (Ref 1, Ref 5, Ref 6, Ref 7).

5. Biomarker and Diagnostic Assays

   • Active MMP-8 (aMMP-8) is measured in clinical samples (e.g., mouth rinse, gingival crevicular fluid) as a biomarker for periodontitis and peri-implantitis, and for monitoring treatment response.
   • Example: Use of MMP-8 assays to differentiate periodontitis from gingivitis and monitor anti-infective therapy (Ref 6).

6. Immunohistochemistry and Histochemical Analysis

   • Recombinant MMP-8 is used in immunohistochemical staining to detect and quantify MMP-8 in tissue sections, particularly in studies of inflammation and disease pathology.
   • Example: Immunohistochemistry in periodontal disease and atheroma-associated cells (Ref 6, Ref 7).

7. Protein-Protein Interaction and Signaling Studies

   • MMP-8 is used to study its interactions with other proteins and its role in signaling pathways, such as in the context of immune cell function and tissue homeostasis.
   • Example: Studies on MMP-8’s role in phagocytosis and immune cell signaling (Ref 1, Ref 5).

These applications highlight the versatility of recombinant human MMP-8 in both basic research and translational studies, spanning enzymatic, cellular, tissue, and whole-organism levels.

Reconstitution of Recombinant Human MMP-8

Recombinant Human MMP-8 is typically supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The protein should be reconstituted to a concentration of 0.1-0.5 mg/mL in sterile distilled water. When reconstituting, centrifuge the tube before opening and avoid vigorous vortexing or pipetting, as this can cause foaming and protein denaturation. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation.

Storage Considerations

After reconstitution, the protein solution remains stable at -20°C for 3 months and at 2-8°C for up to 1 week. To minimize freeze-thaw cycles and maintain protein integrity, it is recommended to aliquot the reconstituted protein solution into smaller portions for individual use. For extended storage, consider adding a carrier protein or stabilizer such as 0.1% bovine serum albumin (BSA), 5% human serum albumin (HSA), 10% fetal bovine serum (FBS), or 5% trehalose.

Activation for Bioassay Applications

An important consideration is that recombinant MMP-8 is supplied in its latent (inactive) form and requires activation before use in functional bioassays. To activate the protein, incubate 100 µg/mL of MMP-8 with 1 mM 4-aminophenylmercuric acetate (APMA) in assay buffer at 37°C for 1 hour. The standard assay buffer composition is 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. After activation, dilute the activated MMP-8 to the desired working concentration (typically 1.0 ng/µL) in assay buffer for your specific experimental application.

Key Preparation Guidelines

• Use sterile, distilled water for reconstitution unless otherwise specified in product documentation
• Avoid repeated freeze-thaw cycles by preparing aliquots
• Handle gently during reconstitution to prevent protein aggregation
• Activate the protein only when performing functional assays requiring enzymatic activity
• Store reconstituted protein at appropriate temperatures based on your experimental timeline