Recombinant Mouse Osteopontin

Using Recombinant Mouse Osteopontin (OPN) in research applications is valuable because OPN is a multifunctional protein involved in key biological processes such as bone remodeling, immune regulation, inflammation, tissue repair, and cancer progression. Recombinant OPN enables controlled, reproducible studies of these mechanisms in vitro and in vivo.

Key reasons to use recombinant mouse OPN include:

• Bone and Mineral Research: OPN inhibits bone mineralization, regulates osteoclast and osteoblast activity, and is essential for studying bone remodeling, osseointegration, and related pathologies.
• Immunology and Inflammation: OPN acts as a cytokine, modulating immune cell migration, macrophage polarization, dendritic cell function, and inflammatory responses. It is implicated in autoimmune diseases, sepsis, and allergic airway disease.
• Cancer Biology: OPN promotes tumor cell proliferation, metastasis, and immune evasion. It is a therapeutic target in breast and lung cancer models, and its inhibition reduces tumor growth and recurrence.
• Tissue Repair and Regeneration: OPN stimulates wound healing, tissue remodeling, and regeneration by activating mesenchymal stem cells and coordinating pro- and anti-inflammatory responses.
• Neurological and Vascular Studies: Recombinant OPN has neuroprotective effects in stroke models, stabilizes vascular smooth muscle phenotype, and improves neurological outcomes after brain injury.
• Foreign Body Response: Recombinant OPN reduces foreign body response, making it useful in biomaterials and implant research.
• Cell Adhesion and Migration: OPN supports cell adhesion, migration, and survival, making it a useful tool in cell biology assays and mechanistic studies.

Experimental applications include:

• Bioassays for cell adhesion, migration, and signaling
• In vivo models of disease (e.g., cancer, stroke, sepsis, wound healing)
• Pull-down assays and mechanistic studies of OPN-receptor interactions
• Functional studies in cell culture and tissue explants

Using recombinant protein ensures batch-to-batch consistency, defined activity, and the ability to dissect OPN’s specific roles in mouse models, which is critical for translational and mechanistic research.

Yes, recombinant mouse osteopontin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in both commercial and custom ELISA protocols.

Supporting details and best practices:

• Commercial ELISA kits for mouse osteopontin routinely use recombinant mouse osteopontin as the standard protein for generating calibration curves. These standards are typically full-length, highly purified recombinant proteins, and their concentrations are precisely determined.
• Assay validation: Published kit protocols and technical datasheets confirm that recombinant osteopontin yields standard curves that are parallel to those generated with natural osteopontin, allowing for accurate quantification of both recombinant and endogenous forms in samples.
• Purity and formulation: For use as an ELISA standard, the recombinant protein should be highly pure (typically >95%) and free from interfering substances such as carrier proteins (unless the assay specifically requires them). Carrier-free formulations are preferred if BSA or other additives could interfere with antibody binding or detection.
• Concentration accuracy: The recombinant standard must have a precisely determined concentration, ideally established by absorbance at 280 nm using a known extinction coefficient or by amino acid analysis. Inaccurate standard concentration will directly affect the accuracy of your quantification.
• Compatibility: Ensure that the recombinant osteopontin you use matches the sequence and post-translational modifications (if relevant) of the target detected by your ELISA antibodies. Most commercial kits are validated to recognize both natural and recombinant forms.
• Documentation: When using a recombinant standard not supplied with a commercial kit, document the source, lot, and concentration determination method for reproducibility and transparency.

Limitations and considerations:

• Matrix effects: If your samples are in a complex matrix (e.g., serum, plasma), ensure that the standard is diluted in a similar matrix or in the same buffer as your samples to minimize matrix effects.
• Assay-specific validation: If you are developing a custom ELISA or using a kit in a non-standard way, validate that your recombinant standard produces a linear, parallel standard curve in your assay conditions.
• Research use only: Most recombinant standards and ELISA kits are for research use only and not for diagnostic purposes.

Summary:
You can use recombinant mouse osteopontin as a standard for ELISA quantification, provided it is highly pure, accurately quantified, and compatible with your assay antibodies and conditions. Always validate the standard curve and ensure parallelism with endogenous osteopontin in your sample matrix.

Recombinant Mouse Osteopontin has been validated in published research for a broad range of applications, primarily in bioassays, in vivo studies, cell culture, and as a functional modulator in disease models.

Key validated applications include:

• Bioassays: Used to assess cell adhesion (e.g., HEK293 cell adhesion), immune cell activation, and functional responses in various cell types.
• In Vivo Assays: Extensively applied in mouse models to study roles in tumor progression, immune modulation, metabolic disorders, neuroprotection (e.g., stroke, brain injury), wound healing, and infection.
• Cell Culture: Supports cell adhesion, differentiation, and functional studies in vitro, including osteogenesis and neurogenesis.
• ELISA Standard/Capture: Used as a standard or capture protein in ELISA assays for quantifying osteopontin or related analytes.
• Positive Control: Serves as a positive control in studies of vascular calcification, atherosclerosis, and hydroxyapatite crystal growth.

Representative published research applications:

• Neuroprotection and Brain Injury: Demonstrated to stabilize vascular smooth muscle cell phenotype and improve neurological outcomes after subarachnoid hemorrhage via intranasal administration in mice.
• Tumor Biology and Immune Modulation: Shown to promote expansion of myeloid-derived suppressor cells, facilitate tumor immune escape, and drive metastatic tumor growth in mouse models.
• Metabolic Disease: Used to study exacerbation of high-fat diet-induced metabolic disorders and age-related adipose tissue remodeling.
• Wound Healing and Regeneration: Validated for recruiting pro-regenerative macrophages and activating mesenchymal stem cells for tissue repair.
• Infection and Inflammation: Applied in models of keratitis and viral neuroinvasion to study immune cell recruitment and inflammatory responses.
• Bone and Dental Research: Used to promote osteogenesis and study bone remodeling and osseointegration on implant surfaces.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant mouse osteopontin in diverse experimental systems.

To reconstitute and prepare Recombinant Mouse Osteopontin protein for cell culture experiments, dissolve the lyophilized protein at a concentration of 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). For carrier-free formulations, reconstitute at 200 μg/mL in sterile PBS.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS to the vial to achieve the recommended concentration (typically 100 μg/mL with carrier protein, or 200 μg/mL for carrier-free).
  • If using a carrier protein, ensure the PBS contains at least 0.1% BSA or human serum albumin (HSA) to stabilize the protein and prevent adsorption to surfaces.
  • Gently mix by pipetting or slow vortexing; avoid vigorous agitation or foaming.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution into small volumes to avoid repeated freeze-thaw cycles.
  • Store aliquots at -20°C to -70°C for long-term storage, or at 2°C to 8°C for short-term use (up to one month).
  • For extended storage, consider adding 5–50% glycerol to the aliquots.

• Preparation for Cell Culture:

  • Thaw aliquots on ice and dilute to the desired working concentration in cell culture medium immediately before use.
  • The effective concentration for cell adhesion assays is typically in the range of 0.1–1.2 μg/mL, but optimal concentrations may vary depending on your specific cell type and experimental design.
  • Avoid repeated freeze-thaw cycles to maintain protein integrity.

Additional best practices:

• Always use sterile technique to prevent contamination.
• If the protein is to be immobilized (e.g., for adhesion assays), coat culture surfaces with the protein at the desired concentration and incubate as per your protocol.
• If you need to further dilute the protein, use buffer containing carrier protein (e.g., 0.1% BSA) to minimize loss due to adsorption.

Summary Table:

Key notes:

• Use a manual defrost freezer for long-term storage.
• Avoid repeated freeze-thaw cycles.
• Always refer to the specific product datasheet for any additional instructions.

This protocol ensures optimal stability and bioactivity of recombinant mouse osteopontin for cell culture applications.