Recombinant Mouse EphB3

Recombinant Mouse EphB3 is a valuable tool for research applications focused on neural development, injury response, cell signaling, and tissue regeneration due to its critical roles in axon guidance, synaptic formation, cell survival, and differentiation.

Key scientific applications and rationale for using recombinant Mouse EphB3 include:

• Neural Injury and Repair Studies
  EphB3 acts as a dependence receptor mediating cell death in oligodendrocytes and neurons after central nervous system (CNS) injury, such as spinal cord injury or traumatic brain injury. Recombinant EphB3 enables mechanistic studies of cell death pathways and therapeutic interventions, such as ligand (ephrinB3) administration to block EphB3-mediated apoptosis and promote cell survival and functional recovery.

• Axon Guidance and Plasticity
  EphB3 is essential for embryonic neuronal axon pathfinding and supports adult axonal growth and plasticity. Recombinant EphB3 can be used in vitro to study axon extension, branching, and regeneration, as well as in vivo models of neural injury and repair.

• Synaptic Formation and Maturation
  EphB3, along with other EphB family members, regulates dendritic spine density and synaptic maturation. Triple knockout studies show abnormal spine formation, highlighting EphB3’s role in synaptic development. Recombinant protein can be used to dissect EphB3-specific signaling in synaptogenesis and plasticity.

• Cell Differentiation and Tissue Regeneration
  EphB3 influences the differentiation and activity of bone-forming cells and neural precursor cells, making it relevant for studies in skeletal development and neurogenesis. Recombinant EphB3 allows for controlled investigation of these processes in cell culture and animal models.

• Signaling Pathway Analysis
  Recombinant EphB3 is suitable for biochemical assays, receptor-ligand binding studies, and pathway elucidation, including kinase-dependent and independent mechanisms affecting cell adhesion and migration.

• Therapeutic Target Validation
  EphB3 is implicated in disease progression and tissue repair, making recombinant protein essential for screening inhibitors, testing ligand-based therapies, and validating targets in preclinical models.

Best practices for using recombinant Mouse EphB3:

• Employ in vitro cell culture systems to study receptor-ligand interactions, apoptosis, and differentiation.
• Use in vivo models to assess functional outcomes of EphB3 modulation in neural injury, regeneration, and development.
• Combine with ephrinB3 ligands or inhibitors to dissect signaling mechanisms and therapeutic potential.

In summary, recombinant Mouse EphB3 is a scientifically robust reagent for dissecting its multifaceted roles in neural, skeletal, and regenerative biology, and for developing targeted therapeutic strategies in CNS injury and related pathologies.

You can use recombinant Mouse EphB3 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with your assay’s antibody pair and detection system.

Key considerations and best practices:

• Purity and Quantification: The recombinant protein should be highly purified, and its concentration should be precisely measured, ideally by HPLC or another validated method. Impurities or inaccurate quantification can lead to errors in your standard curve and subsequent sample quantification.

• Standard Curve Preparation: Recombinant proteins are commonly used to generate standard curves in ELISA, especially when native protein standards are unavailable. Prepare serial dilutions of the recombinant EphB3 to cover the expected concentration range of your samples.

• Assay Compatibility: Ensure that the recombinant EphB3 is recognized by the capture and detection antibodies in your ELISA. Some kits are validated specifically with certain recombinant forms (e.g., His-tagged, Fc-chimera), so check that your protein’s tag or modifications do not interfere with antibody binding.

• Matrix Effects: For best accuracy, dilute your recombinant standard in the same matrix as your samples (e.g., serum, plasma, or buffer) to minimize matrix effects and improve comparability.

• Validation: It is good practice to validate the use of your recombinant standard by comparing the standard curve generated with it to that obtained using a reference standard, if available, or by spike-and-recovery experiments in your sample matrix.

• Documentation: Some ELISA kits and protocols specifically mention using recombinant proteins as standards, and many commercial kits use recombinant EphB3 as the calibrator.

Limitations:

• Quantification using recombinant standards may not always reflect the exact concentration of endogenous protein due to differences in post-translational modifications or folding between recombinant and native forms.
• Recovery rates can vary, and a ±25% difference from the stated amount is not uncommon when using recombinant proteins as standards in ELISA.

Summary Table: Recombinant Protein as ELISA Standard

In summary, recombinant Mouse EphB3 is suitable as a standard for ELISA quantification if you follow best practices for preparation, validation, and assay optimization.

Recombinant Mouse EphB3 has been validated for several applications in published research, primarily in studies of receptor-ligand interactions, cell signaling, viral entry, and functional assays.

Key validated applications include:

• Enzyme kinetics and inhibitor screening: Recombinant EphB3 has been used to study enzyme kinetics, screen for inhibitors, and perform selectivity profiling, particularly in the context of kinase activity and drug discovery.
• Cell signaling and functional assays: It has been applied in cell-based assays to investigate EphB3-mediated signaling pathways, including its role in osteogenesis, angiogenesis, and cell death regulation.
• Viral entry studies: Soluble recombinant mouse EphB3 has been used as a decoy receptor to block viral infection (e.g., murine gammaherpesvirus 68), demonstrating its utility in receptor-ligand competition and viral entry assays.
• Protein-protein interaction studies: Recombinant EphB3 is commonly used in binding assays to characterize interactions with ephrin ligands and other proteins, including Fc chimera formats for enhanced detection and quantification.
• Biochemical and biophysical characterization: It has been used for structural and functional studies, including analysis of kinase activity, dimerization, and receptor clustering.
• In vivo functional rescue experiments: While not directly using recombinant EphB3, related studies have used recombinant ephrinB3 to modulate EphB3 signaling in animal models, highlighting the pathway's relevance in neurovascular injury and repair.

Additional applications, as indicated by product descriptions and research use statements, include:

• Use as a standard in ELISA and other immunoassays.
• Use in cell culture experiments to probe EphB3 function or as a ligand for ephrin-expressing cells.

These applications are supported by published research and product documentation, demonstrating the versatility of recombinant mouse EphB3 in molecular, cellular, and biochemical studies.

To reconstitute and prepare Recombinant Mouse EphB3 protein for cell culture experiments, add sterile water or buffer to achieve a concentration between 0.1–1.0 mg/mL, then aliquot and store under sterile conditions to avoid repeated freeze-thaw cycles.

Detailed protocol and best practices:

• Centrifuge the vial briefly at 4°C before opening to ensure all lyophilized protein is at the bottom.
• Reconstitution:
  • Add sterile, endotoxin-free water or sterile PBS to the vial. The recommended concentration is typically 0.1–1.0 mg/mL.
  • For example, to prepare a 0.2 µg/µL (200 µg/mL) stock, add the appropriate volume of water as indicated on the product datasheet.
  • Gently pipette up and down or swirl to dissolve. Avoid vigorous vortexing to prevent protein denaturation.
• Optional additives:
  • For enhanced stability, especially for long-term storage or repeated freeze-thaw, consider adding carrier protein (e.g., 0.1% BSA) or glycerol (final 5–50%) to the solution.
• Aliquoting and storage:
  • Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C to –80°C for long-term use, or at 4°C for short-term use (up to 1 week).
  • Avoid repeated freeze-thaw cycles, as this can reduce protein activity and integrity.
• Sterility:
  • Always use sterile technique and solutions to prevent contamination, especially for cell culture applications.
• Working solution:
  • Before adding to cell cultures, dilute the stock solution to the desired working concentration using sterile cell culture medium or buffer compatible with your assay.

Additional notes:

• Always consult the specific product datasheet for any protein-specific recommendations, as some preparations may require a particular buffer or have unique stability considerations.
• If the protein is to be used for sensitive cell types or in vivo, ensure the endotoxin level is sufficiently low.

Summary table:

Following these steps will help maintain the biological activity and integrity of recombinant Mouse EphB3 protein for reliable cell culture experiments.