Recombinant Human MMP-13

Recombinant Human MMP-13 (Matrix Metalloproteinase-13) is a valuable tool for a wide range of research applications due to its well-characterized enzymatic activity and biological relevance. Here are several key reasons why you should consider using recombinant human MMP-13 in your research:

1. Controlled and Reproducible Enzyme Activity

Recombinant MMP-13 is produced under standardized conditions, ensuring batch-to-batch consistency and high purity. This allows for reproducible experimental results, which is critical for enzyme kinetics, substrate cleavage studies, and functional assays.

2. Versatile Applications

Recombinant MMP-13 is useful for:

• Studying enzyme regulation and kinetics
• Cleaving specific substrates in vitro or in cell culture
• Serving as a positive control in zymography or immunoblotting
• Investigating the role of MMP-13 in extracellular matrix (ECM) remodeling

3. Biological Relevance

MMP-13 (also known as Collagenase-3) is a major interstitial collagenase that degrades a broad range of ECM proteins, including collagen types I, II, III, IV, IX, X, and XIV. Its activity is central to processes such as:

• Tissue remodeling
• Wound healing
• Angiogenesis
• Inflammation
• Cancer progression and metastasis

4. Functional Studies in Disease Models

MMP-13 has been implicated in various pathological conditions, including:

• Arthritis and joint diseases
• Cancer invasion and metastasis
• Atherosclerosis
• Fibrosis
• Sepsis

Using recombinant MMP-13 allows you to directly assess its effects on cell behavior, signaling pathways, and disease progression in controlled experimental settings.

5. Ease of Use

Recombinant MMP-13 is typically supplied in a lyophilized or liquid form that is easy to dissolve, aliquot, and use without loss of enzymatic activity. This practicality makes it suitable for both short-term and long-term experiments.

6. Mechanistic Insights

By adding recombinant MMP-13 to cell cultures or tissue models, you can:

• Induce specific ECM degradation
• Study downstream signaling events
• Investigate the role of MMP-13 in inflammation, angiogenesis, or fibrosis
• Validate the effects of MMP-13 inhibitors or activators

7. Support for Drug Discovery and Therapeutic Development

Recombinant MMP-13 is essential for screening and validating potential inhibitors or activators, making it a key reagent in drug discovery pipelines targeting MMP-13-related diseases.

In summary, recombinant human MMP-13 is a reliable, versatile, and biologically relevant reagent that enables precise investigation of MMP-13 function in both physiological and pathological contexts. Its use can significantly enhance the rigor and impact of your research.

Using Recombinant Human MMP-13 as ELISA Standards

Recombinant human MMP-13 can be used as a standard for quantification and calibration in ELISA assays, but with important considerations regarding the specific application and assay design.

Appropriate Applications

Recombinant MMP-13 is well-suited for use as a standard in sandwich ELISA formats designed to measure the protein. The recombinant protein can be diluted to appropriate concentrations in assay buffer to establish standard curves for quantification. This approach is particularly effective when the ELISA kit is specifically designed to recognize and measure MMP-13 protein levels in various sample types including serum, plasma, cell culture supernatants, and tissue lysates.

Critical Limitation: Detection of Recombinant Protein

However, there is a significant restriction to be aware of: recombinant MMP-13 should not be used for direct detection within certain ELISA kit formats. Some commercial ELISA kits are specifically optimized to detect endogenous MMP-13 in biological samples rather than recombinant protein, and using recombinant protein directly in these assays is not recommended.

Best Practices for Standard Preparation

When using recombinant MMP-13 as a standard, follow these guidelines:

Preparation and Storage: Store the recombinant protein at ≤-20°C and prepare working standards fresh for each assay run. Standard solutions are best used within 2 hours of preparation.

Activation: If necessary for your application, recombinant MMP-13 can be activated by adding APMA (4-aminophenylmercuric acetate) to a final concentration of 1 mM in assay buffer.

Standard Curve Establishment: A standard curve must be established for every assay run, as exact conditions may vary between experiments. Typical standard curves range from approximately 6-6000 pg/mL depending on the specific assay format.

Verification of Suitability

Before committing to recombinant MMP-13 as your standard, verify that your ELISA kit is compatible with recombinant protein standards. Confirm that the kit demonstrates appropriate recovery rates and linearity when recombinant standards are used, and ensure there is no cross-reactivity with other matrix metalloproteinases or tissue inhibitors of metalloproteinases (TIMPs) that might be present in your samples.

Recombinant Human MMP-13 has been validated for a range of applications in published research, including bioassays, cell and tissue culture, Western blotting, enzyme activity assays, substrate cleavage studies, and as a control in inflammation models.

Key validated applications include:

• Bioassays: Used to assess enzymatic activity, substrate specificity, and inhibitor screening, particularly in studies of arthritis and cartilage degradation.
• Cell and Tissue Culture: Applied to study MMP-13’s effects on cell behavior, extracellular matrix remodeling, and inflammatory responses in various cell types, such as chondrocytes, osteoblast-like cells, and macrophages.
• Western Blotting: Utilized as a positive control for protein detection and quantification in immunoblotting experiments.
• Enzyme Activity Assays: Employed in fluorogenic and colorimetric assays to measure proteolytic activity, including collagen zymography for quantifying collagenase activity.
• Substrate Cleavage Studies: Used to investigate cleavage of extracellular matrix proteins (e.g., collagen types I, II, III, IV) and to validate the specificity of MMP-13 for various substrates in vitro.
• Inflammation and Disease Models: Validated as a tool to induce or modulate inflammation in cell-based models (e.g., THP-1 monocyte-derived macrophages), and in animal models of rheumatoid arthritis and tissue fibrosis.
• ELISA Standard: Used as a standard for quantification in ELISA assays.

Additional context:

• Recombinant MMP-13 is frequently used to study matrix remodeling, joint diseases (especially arthritis), wound healing, and cancer due to its role in collagen degradation and tissue invasion.
• It serves as a control or reference enzyme in studies evaluating the efficacy of MMP-13 inhibitors and in mechanistic investigations of extracellular matrix turnover.

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the protein’s utility in both basic and translational research settings.

To reconstitute and prepare Recombinant Human MMP-13 protein for cell culture experiments, follow these general best practices and specific recommendations based on manufacturer protocols and scientific literature:

1. Reconstitution

• Centrifuge the vial before opening to ensure all lyophilized protein is at the bottom.
• Reconstitute with sterile, endotoxin-free water or an appropriate buffer (e.g., sterile PBS or Tris buffer, pH 7.4–7.6). Most manufacturers recommend sterile distilled water unless otherwise specified.
• Typical reconstitution concentration: 0.1–0.5 mg/mL (check the product datasheet for exact recommendations).
• Gently mix by swirling or tapping the vial; avoid vortexing or vigorous pipetting to prevent protein denaturation.
• Allow the protein to dissolve at room temperature for 10–15 minutes, or gently rotate at 4°C for 30 minutes if needed.

2. Storage of Reconstituted Protein

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –80°C for long-term storage (typically stable for 6–12 months).
• For short-term use (within a few days), store at 2–8°C.
• Use polypropylene tubes for storage to minimize protein adsorption.

3. Activation (if required)

• MMP-13 is often supplied as a pro-enzyme (latent form) and may require activation for full enzymatic activity.
• Activation protocol (commonly used):
  • Dilute the reconstituted protein in assay buffer (e.g., 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5).
  • Add 1 mM APMA (4-aminophenylmercuric acetate) to activate the enzyme.
  • Incubate at 37°C for 1–2 hours.
  • After activation, dilute to the desired working concentration in cell culture medium or assay buffer.

4. Use in Cell Culture

• Dilute activated MMP-13 in serum-free or low-serum cell culture medium to the desired concentration (typically in the ng/mL to µg/mL range, depending on the experiment).
• Include appropriate controls (e.g., vehicle-only, inactive enzyme).
• Add the protein directly to the culture medium and incubate cells for the required time.
• Monitor cell viability and morphology during treatment.

5. Additional Tips

• Avoid repeated freeze-thaw cycles of reconstituted protein.
• Use sterile techniques throughout to prevent contamination.
• Check the datasheet for lot-specific instructions and recommended buffer compositions.

References

• Manufacturer protocols (e.g., Abcam, R&D Systems, Bio-Techne, Leinco, MedchemExpress, Assay Genie).
• General recombinant protein handling guidelines (Thermo Fisher, ABclonal).

If you have a specific product code or manufacturer, consult the product datasheet for exact reconstitution and activation instructions.