Recombinant Mouse Osteoprotegerin

Recombinant Mouse Osteoprotegerin (OPG) is a valuable tool in research for studying bone metabolism, osteoclast regulation, and the interplay between bone and cancer, due to its potent ability to inhibit osteoclastogenesis and modulate RANKL signaling.

Key scientific applications and rationale include:

• Bone Metabolism and Osteoclast Inhibition: OPG acts as a decoy receptor for RANKL, preventing RANKL from binding to its receptor RANK on osteoclast precursors, thereby inhibiting osteoclast differentiation and activity. This mechanism is central to studies on osteoporosis, osteopetrosis, and bone remodeling. Recombinant OPG has been shown to reverse osteoporosis in OPG-deficient mice and increase bone density by reducing bone resorption.

• Cancer Research and Bone Metastasis: OPG is involved in the regulation of bone metastases in cancers such as breast and multiple myeloma. Recombinant OPG can suppress tumor-induced bone destruction, inhibit tumor growth, and reduce metastasis by interfering with the RANK/RANKL pathway. It also modulates cancer cell behavior, including epithelial-to-mesenchymal transition (EMT), stemness, and migration.

• Immunomodulation and Inflammation: By blocking RANKL, OPG influences immune cell differentiation, particularly regulatory T cells, and can be used to study immune responses in autoimmune diseases and inflammatory bone loss.

• Orthodontics and Bone Healing: Recombinant OPG has been used experimentally to enhance bone quantity and anchorage during orthodontic tooth movement by inhibiting osteoclastogenesis, making it relevant for dental and craniofacial research.

• Translational and Therapeutic Studies: Recombinant OPG constructs have been evaluated in preclinical and early clinical studies as potential therapeutics for bone diseases and cancer-related bone loss, providing a model for drug development and mechanistic studies.

Summary of best practices:

• Use recombinant mouse OPG in vitro to study osteoclast differentiation, bone resorption assays, and RANKL signaling.
• Employ in vivo mouse models to investigate bone density, osteoporosis, osteopetrosis, and cancer metastasis to bone.
• Consider OPG’s dual role in tumor biology—both as a suppressor and promoter depending on context—when interpreting results.

In summary, recombinant mouse OPG is a critical reagent for dissecting the molecular mechanisms of bone remodeling, cancer metastasis, and immune regulation, and for developing new therapeutic strategies targeting the RANK/RANKL/OPG axis.

You can use recombinant Mouse Osteoprotegerin (OPG) as a standard for quantification or calibration in ELISA assays, provided the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with the antibodies and detection system used in your assay.

Supporting details:

• Multiple commercial ELISA kits for mouse OPG use recombinant mouse OPG as the standard for generating calibration curves, and these kits have been validated to accurately quantify both recombinant and endogenous OPG in biological samples.
• Product datasheets and technical documents explicitly state that their immunoassays "have been shown to quantitate recombinant mouse OPG accurately" and that "results obtained using natural mouse OPG showed dose response curves that were parallel to the standard curves obtained using the recombinant protein," indicating equivalence in assay performance.
• When using recombinant OPG as a standard, ensure that:
  • The recombinant protein is in a buffer compatible with your assay diluent.
  • The protein is fully soluble and not aggregated.
  • The concentration is verified by an orthogonal method (e.g., absorbance at 280 nm, BCA assay).
  • The standard curve covers the expected range of OPG concentrations in your samples.
• Some ELISA kits may be optimized for detection of endogenous OPG and may not be validated for recombinant forms with different tags or modifications; always check the kit documentation for compatibility.

Best practices:

• Prepare serial dilutions of the recombinant OPG in the same matrix as your samples (e.g., assay buffer, serum, or plasma) to minimize matrix effects.
• Validate the linearity and recovery of the recombinant standard in your specific assay setup, especially if using a custom or in-house ELISA protocol.
• If using a recombinant OPG-Fc chimera or other fusion constructs, confirm that the epitopes recognized by the capture and detection antibodies are present and accessible.

Limitations:

• Do not use recombinant OPG as a standard if the ELISA kit documentation specifically advises against it or if the recombinant protein differs significantly in structure or post-translational modifications from the endogenous protein in your samples.

In summary, recombinant mouse OPG is widely used and accepted as a standard for ELISA quantification, but always confirm compatibility with your specific assay system and validate performance as needed.

Recombinant Mouse Osteoprotegerin (OPG) has been validated for several key applications in published research, primarily in studies involving bone metabolism, cancer, diabetes, vascular biology, and fibrogenesis.

Validated Applications in Published Research:

• In vivo therapeutic studies:
  Recombinant mouse OPG has been administered to mouse models to investigate its effects on bone resorption, bone metastasis, and tumor progression. It has demonstrated efficacy in reducing tumor burden, delaying tumor onset, and preventing bone damage in models of multiple myeloma, breast cancer, prostate cancer, and non-small cell lung cancer.

• Diabetes and β-cell biology:
  OPG has been used to stimulate β-cell proliferation in young, aged, and diabetic mice, resulting in increased β-cell mass and delayed hyperglycemia. It has also been validated for stimulating human β-cell replication in vitro and in vivo (humanized mice), with mechanistic studies showing modulation of CREB and GSK3 pathways.

• Vascular and cardiovascular research:
  Recombinant OPG has been tested in mouse models of abdominal aortic aneurysm (AAA) to assess its effects on disease development. It has also been used in studies of endothelial cell adhesion, vascular smooth muscle cell apoptosis, and matrix remodeling relevant to atherosclerosis.

• Fibrogenesis and biomarker studies:
  OPG has been linked to fibrogenesis in various tissues, including the liver, and is being evaluated as a non-invasive biomarker for fibrotic diseases.

• In vitro functional assays:
  Recombinant mouse OPG has been validated for use in cell-based assays, such as inhibition of TRAIL-mediated cytotoxicity in mouse fibrosarcoma cells, and for studying osteoclastogenesis and bone resorption mechanisms.

• Preclinical therapeutic evaluations:
  OPG is widely used as a reagent in mouse model studies for comparative immunology and preclinical therapeutic research, including evaluation of its effects on bone and immune cell development.

Common Experimental Protocols:

• In vivo administration:
  Intraperitoneal or intravenous injection of recombinant OPG in mouse models to study bone, cancer, diabetes, and vascular outcomes.

• In vitro cell culture:
  Addition of recombinant OPG to cell cultures to assess effects on cell proliferation, apoptosis, differentiation, and signaling pathways.

• Biomarker quantification:
  Use as a standard or control in ELISA and Western blot assays for quantifying OPG levels in biological samples.

Summary Table of Validated Applications

Key Insights:

• Recombinant mouse OPG is a versatile tool validated for both in vivo and in vitro studies across multiple disease models.
• Its primary scientific applications are in bone biology, cancer metastasis, diabetes, vascular disease, and fibrogenesis.
• Protocols typically involve direct administration to mice or addition to cell cultures, with functional readouts including proliferation, apoptosis, and biomarker quantification.

If you require protocols for a specific application or further details on experimental design, please specify the research context.

To reconstitute Recombinant Mouse Osteoprotegerin protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer—commonly sterile PBS or distilled water—to a concentration of at least 100 μg/mL. After reconstitution, gently mix and allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.

Detailed protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the powder at the bottom.
  • Use sterile technique throughout to avoid contamination.

• Reconstitution:

  • Add sterile PBS (pH 7.4) or sterile distilled water to achieve a final concentration of 100 μg/mL or higher.
  • Gently mix by pipetting or slow inversion; avoid vigorous shaking to prevent foaming and protein denaturation.
  • Let the solution stand at room temperature for 15–30 minutes to ensure full dissolution.

• Aliquoting and Storage:

  • If not used immediately, aliquot the solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at 4°C for short-term use (2–7 days) or at –20°C to –70°C for long-term storage.
  • For extended storage, adding a carrier protein such as 0.1% BSA or HSA is recommended to stabilize the protein.
  • Avoid repeated freeze-thaw cycles, as this can degrade the protein.

• Preparation for Cell Culture:

  • Before use, dilute the stock solution into cell culture medium to the desired working concentration.
  • Ensure the final buffer composition is compatible with your cell culture system (e.g., serum-free or serum-containing medium).

Additional notes:

• Always consult the specific product datasheet for any unique requirements regarding buffer composition or concentration.
• Recombinant proteins produced in E. coli are not glycosylated, which may affect stability but generally not in vitro activity.
• For in vivo studies, glycosylated forms (from mammalian or insect cells) may be preferable due to increased stability.

This protocol ensures optimal solubility and stability of recombinant mouse osteoprotegerin for cell culture applications.