Recombinant Mouse MMP-9

Recombinant mouse MMP-9 (matrix metalloproteinase-9, also known as gelatinase B) is a valuable tool for research applications due to its diverse biological functions and well-characterized enzymatic properties.

Substrate Degradation Capabilities

Recombinant mouse MMP-9 possesses broad substrate specificity, making it useful for studying extracellular matrix remodeling. The protein can degrade gelatin, collagen types IV and V, elastin, and proteoglycan core protein. This enzymatic versatility allows researchers to investigate matrix degradation processes relevant to various physiological and pathological conditions.

Cytokine and Chemokine Processing

MMP-9 plays an important role in processing signaling molecules. Notably, it releases the biologically active form of vascular endothelial growth factor (VEGF), suggesting its involvement in angiogenesis and new blood vessel formation. This capability makes recombinant MMP-9 particularly useful for studying growth factor activation and vascular biology.

Immune Cell Migration Studies

The protein participates in immune cell migration, as demonstrated through studies using MMP-9 knockout mice following antigen challenge. Researchers can use recombinant MMP-9 to investigate mechanisms of immune cell recruitment and infiltration in various inflammatory contexts.

Receptor Signaling Regulation

MMP-9 regulates the activity of multiple transmembrane receptors, including nerve growth factor (NGF)-induced tyrosine kinase A (TrkA), EGFR/HER, and insulin receptor (IR) signaling. This makes it valuable for studying signal transduction pathways and receptor biology in cancer and other disease models.

Disease Model Applications

Recombinant mouse MMP-9 is applicable to research involving tumorigenesis, cardiovascular disease, wound healing, and inflammatory conditions. The protein's role in epithelial-to-mesenchymal transition, cell migration, and immune response regulation makes it relevant for investigating disease mechanisms and therapeutic targets.

Experimental Flexibility

Using recombinant MMP-9 provides experimental control and reproducibility compared to endogenous sources. The carrier-free formulation available for research allows for precise dosing and standardized protocols across experiments, facilitating reliable bioassay development, Western blot analysis, and ELISA applications.

Yes, recombinant Mouse MMP-9 can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay's antibodies and detection system. Recombinant proteins are commonly supplied as standards in commercial mouse MMP-9 ELISA kits, and their use is well-established for generating standard curves to quantify MMP-9 concentrations in biological samples.

Essential context and best practices:

• Assay Compatibility: Ensure that the recombinant MMP-9 standard is recognized by the capture and detection antibodies in your ELISA. Most sandwich ELISA kits for mouse MMP-9 are designed to detect both native and recombinant forms, but you should confirm this in your kit's documentation or by consulting the antibody specificity.
• Standard Preparation: Recombinant MMP-9 is typically supplied lyophilized and should be reconstituted and diluted according to the kit protocol to generate a standard curve covering the assay's dynamic range.
• Matrix Effects: When quantifying MMP-9 in complex biological samples (e.g., serum, plasma, tissue lysates), matrix components may affect detection. It is recommended to prepare standards in a matrix similar to your samples or use appropriate diluents to minimize matrix effects.
• Quality Control: Use internal controls and spike-recovery experiments to validate the accuracy and precision of quantification when using recombinant standards.

Additional relevant information:

• Detection Range: ELISA kits for mouse MMP-9 typically have a detection range from tens to thousands of pg/mL, and recombinant standards are used to interpolate sample concentrations from the standard curve.
• Storage and Stability: Recombinant MMP-9 standards should be stored as recommended (often at -20°C or 4°C) and used within the specified time after reconstitution to ensure stability and accuracy.
• Bioactivity: While recombinant MMP-9 is suitable for quantification, it may not always reflect the bioactivity or post-translational modifications of endogenous MMP-9. For calibration, this is generally not a concern, but for functional assays, native protein may be preferred.

Summary Table: Recombinant Mouse MMP-9 as ELISA Standard

In conclusion: Recombinant Mouse MMP-9 is widely used as a standard for ELISA quantification, but always verify compatibility with your specific assay and validate with appropriate controls.

Recombinant Mouse MMP-9 has been validated for a broad range of applications in published research, including bioassays, enzyme assays, ELISA (as a standard), Western blot, zymography, cell culture, and in vivo studies.

Key validated applications and supporting details:

• Bioassay: Used to assess enzymatic activity, substrate specificity, and inhibitor screening in cell-based and biochemical assays.
• Enzyme Assay: Applied to measure gelatinase activity and to study the proteolytic processing of extracellular matrix proteins.
• ELISA (Standard): Utilized as a quantitative standard for measuring MMP-9 levels in biological samples.
• Western Blot: Employed to detect MMP-9 protein and to validate antibody specificity in cell lysates and tissue homogenates.
• Zymography: Used to analyze gelatinase activity in biological samples, distinguishing active and pro-enzyme forms.
• Cell Culture: Added to cultured cells to study effects on migration, invasion, and matrix remodeling, particularly in cancer and immune cell models.
• In Vivo Studies: Administered in animal models to investigate roles in tissue remodeling, inflammation, angiogenesis, and disease processes such as cerebral ischemia, wound healing, and neovascularization.

Representative research applications:

• Blood-brain barrier studies: Investigating astroglial barrier function and substrate identification using secretomics and recombinant MMP-9.
• Ischemic injury: Assessing neuroprotection and MMP-9 inhibition in mouse models of cerebral ischemia.
• Viral pathogenesis: Studying Zika virus invasion and blood-testis barrier integrity.
• Cancer research: Evaluating cell migration, invasion, and metastasis in thyroid and breast cancer cell lines.
• Wound healing: Exploring diabetic wound healing and the effects of MMP-9 inhibition or modulation in mouse models.
• Angiogenesis and neovascularization: Analyzing MMP-9’s role in VEGF release, vascular remodeling, and ocular neovascularization.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, demonstrating the versatility of recombinant mouse MMP-9 in both basic and translational research.

To reconstitute and prepare Recombinant Mouse MMP-9 protein for cell culture experiments, follow these general guidelines based on standard protocols and manufacturer recommendations:

1. Reconstitution

• Reconstitution Buffer: Use sterile water or a buffer as specified by the manufacturer (often sterile PBS or assay buffer).
• Concentration: Prepare a stock solution at the recommended concentration (commonly 0.25 mg/mL or as indicated on the Certificate of Analysis).
• Procedure: Gently add the appropriate volume of sterile water or buffer to the lyophilized protein. Avoid vigorous mixing or foaming to prevent protein denaturation.
• Storage: Aliquot the reconstituted protein and store at -20°C to -80°C. Avoid repeated freeze-thaw cycles.

2. Activation (if required)

• Activation Buffer: Dilute the reconstituted MMP-9 to 100 µg/mL in assay buffer (typically 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5).
• Activator: Add APMA (4-aminophenylmercuric acetate) to a final concentration of 1 mM.
• Incubation: Incubate at 37°C for 2 hours to activate the protein.
• Dilution: After activation, dilute the MMP-9 to the desired working concentration (e.g., 0.4 ng/µL or as needed for your experiment) in assay buffer.

3. Application in Cell Culture

• Working Concentration: Use the activated and diluted MMP-9 at the appropriate concentration for your cell culture experiment. Typical concentrations range from 0.1 to 10 ng/mL, depending on the experimental design.
• Addition to Cells: Add the MMP-9 solution directly to the cell culture medium. Ensure thorough mixing and incubate the cells under standard culture conditions.

4. General Tips

• Sterility: Maintain sterile conditions throughout the reconstitution and preparation process.
• Buffer Compatibility: Ensure that the buffer used for reconstitution and dilution is compatible with your cell culture system.
• Activity Assay: If required, perform an activity assay to confirm the functionality of the reconstituted and activated MMP-9.

Example Protocol

1. Reconstitute lyophilized MMP-9 in sterile water to 0.25 mg/mL.
2. Aliquot and store at -80°C.
3. For use, dilute to 100 µg/mL in assay buffer.
4. Add APMA to 1 mM final concentration.
5. Incubate at 37°C for 2 hours.
6. Dilute to 0.4 ng/µL in assay buffer.
7. Add to cell culture medium at the desired concentration.

Always refer to the specific product datasheet and Certificate of Analysis for detailed instructions and any unique requirements for the particular batch of recombinant MMP-9 you are using.