Recombinant Human MMP-2

Recombinant Human MMP-2 is widely used in research applications to study extracellular matrix remodeling, tumor invasion, angiogenesis, inflammation, and stem cell migration due to its well-characterized enzymatic activity and relevance in human physiology and pathology.

Key scientific reasons to use Recombinant Human MMP-2:

• Controlled Enzymatic Activity: Recombinant MMP-2 provides a consistent and defined source of enzyme for in vitro assays, allowing precise quantification of substrate cleavage, inhibitor screening, and kinetic studies.
• Extracellular Matrix (ECM) Remodeling: MMP-2 specifically cleaves gelatin and various types of collagen (I, IV, V, VII, X), making it essential for investigating ECM degradation, tissue repair, and fibrosis mechanisms.
• Cancer Research: Elevated MMP-2 activity is linked to tumor invasion, metastasis, and angiogenesis. Recombinant MMP-2 is used to model these processes, test inhibitors, and develop imaging probes for aggressive tumors.
• Inflammation and Vascular Biology: MMP-2 modulates inflammatory pathways by processing cytokines (e.g., TNF-α, IL-1β) and vasoactive molecules, making it valuable for studies on cardiovascular disease, atherosclerosis, and vascular remodeling.
• Stem Cell Migration: MMP-2 is critical for the invasive capacity and migratory behavior of mesenchymal stem cells and other cell types, relevant for regenerative medicine and cell therapy research.
• Assay Development: Recombinant MMP-2 serves as a positive control in zymography, enzyme assays, western blotting, and mass spectrometry, ensuring assay reliability and reproducibility.

Typical applications include:

• Enzyme activity assays and inhibitor screening
• ECM degradation and remodeling studies
• Cancer cell invasion and metastasis models
• Angiogenesis assays
• Inflammatory signaling pathway analysis
• Stem cell migration and tropism studies
• Positive controls for protein detection techniques

Using recombinant human MMP-2 ensures experimental reproducibility, eliminates variability from tissue-derived enzymes, and allows for mechanistic studies relevant to human biology and disease.

Yes, recombinant human MMP-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay's antibody pair and detection system. This is a common practice in quantitative ELISA protocols for MMP-2, as most commercial ELISA kits use recombinant MMP-2 as their calibration standard.

Key considerations and supporting details:

• Standard Curve Generation: Quantitative ELISA kits for human MMP-2 typically include a recombinant human MMP-2 standard to generate a standard curve, which is then used to determine the concentration of MMP-2 in unknown samples. The recombinant standard is serially diluted to cover the assay’s dynamic range.

• Compatibility: The recombinant MMP-2 used as a standard should match the form (e.g., pro, active, or total MMP-2) that your ELISA detects. Most sandwich ELISAs for MMP-2 are designed to recognize both recombinant and native forms, as well as different activation states, but you should confirm this in your assay documentation.

• Validation: It is important to ensure that the recombinant MMP-2 standard you use is recognized by the capture and detection antibodies in your specific ELISA. Commercial kits validate their standards for this purpose, but if you are assembling a custom assay, you should verify cross-reactivity and parallelism between the standard curve and endogenous MMP-2 in your sample matrix.

• Preparation: Follow the manufacturer’s instructions for reconstitution and dilution of the recombinant standard to ensure accurate calibration.

• Best Practices:

  • Use the same buffer and matrix for standard dilutions as for your samples to minimize matrix effects.
  • Confirm that the recombinant protein is of high purity and properly quantified.

Summary Table: Use of Recombinant Human MMP-2 as ELISA Standard

In summary: Using recombinant human MMP-2 as a standard is standard practice in ELISA quantification, but always confirm compatibility with your specific assay system and follow best practices for standard preparation and validation.

Recombinant Human MMP-2 has been validated for a diverse range of applications across published research, reflecting its importance as a tool for studying matrix metalloproteinase biology and function.

Primary Research Applications

Bioassay represents the most extensively documented application, with recombinant MMP-2 used to assess enzymatic activity and protein function in various biological contexts. The protein demonstrates robust catalytic activity, cleaving modified fluorogenic peptide substrates (Mca-PLGL-Dpa-AR-NH2) with specific activities exceeding 1100 pmol/min/µg.

Enzyme Assay applications have been employed to characterize MMP-2 kinetics and substrate specificity. These studies utilize recombinant MMP-2 to measure proteolytic activity against various substrates and to evaluate enzyme inhibition.

Zymography has been used to detect and quantify MMP-2 activity in complex biological samples, including cell culture supernatants and tissue extracts. This technique allows for both qualitative and semi-quantitative analysis of gelatinase activity.

Specialized Applications

Functional Assays and Blocking Assays represent validated applications for studying MMP-2 biological activity and protein-protein interactions. These approaches enable researchers to investigate the role of MMP-2 in cellular processes such as migration, invasion, and extracellular matrix remodeling.

ELISA Standards utilize recombinant MMP-2 as a positive control and reference standard for quantitative immunoassays.

Western Blot and Mass Spectrometry applications employ recombinant MMP-2 as a control protein for validating detection methods and protein identification.

Research Context

The published literature demonstrates MMP-2 application in investigating its roles in angiogenesis, tumor invasion, tissue repair, vascular remodeling, inflammation, and atherosclerotic plaque pathology. Additionally, emerging research has identified novel functions including histone proteolysis and myogenic gene regulation.

To reconstitute and prepare Recombinant Human MMP-2 protein for cell culture experiments, follow these best-practice steps based on current protocols and technical recommendations:

1. Preparation Before Reconstitution

• Centrifuge the vial briefly (20–30 seconds) to collect all lyophilized protein at the bottom and avoid loss when opening.
• Warm the vial to room temperature before opening to prevent condensation.

2. Reconstitution

• Use sterile, distilled water or the buffer recommended in the product datasheet. Most protocols suggest reconstituting to a concentration between 0.1–1.0 mg/mL.
• For example, to achieve 0.1 mg/mL, add 1 mL of sterile water to 0.1 mg of protein.
• Gently mix by swirling or tapping; do not vortex, as vigorous mixing can denature the protein.
• If the protein appears as a film, ensure it is fully dissolved by gentle mixing.
• Allow to stand at room temperature for 15–30 minutes to ensure complete dissolution.

3. Handling and Storage

• Avoid repeated freeze-thaw cycles; aliquot the reconstituted protein if multiple uses are anticipated.
• Store reconstituted protein at –20°C or –70°C (depending on manufacturer’s guidance) and use within 3 months.
• If a precipitate forms, centrifuge briefly before use.

4. Activation (if required)

• Recombinant MMP-2 is often supplied as a proenzyme (inactive form). For enzymatic activity, activate with APMA (p-aminophenylmercuric acetate):
  • Dilute MMP-2 to 100 µg/mL in assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij 35, pH 7.5.
  • Add APMA to a final concentration of 1 mM.
  • Incubate at 37°C for 1 hour to achieve activation.
• For cell culture applications where you want to study the effect of the proenzyme, omit the activation step.

5. Application in Cell Culture

• Dilute the reconstituted (and, if needed, activated) protein into your cell culture medium to the desired working concentration, ensuring compatibility with your experimental design.
• Confirm the absence of cytotoxic additives (e.g., high concentrations of DMSO or APMA) in the final cell culture medium.

6. Quality Control

• Confirm protein integrity and concentration by SDS-PAGE or another suitable method before use.

Summary Table: Key Steps for Recombinant Human MMP-2 Preparation

Note: Always consult the specific product datasheet for any unique requirements, as buffer composition and reconstitution volumes may vary between suppliers.

If you need a protocol tailored to a specific cell type or application (e.g., invasion assays, zymography), please specify for more detailed guidance.