Recombinant Human Ephrin-B3

Using Recombinant Human Ephrin-B3 in research applications is valuable for studying cell signaling, neural development, synaptic regulation, immune cell migration, and cancer biology due to its well-characterized roles as a ligand for Eph receptors and its involvement in multiple physiological and pathological processes.

Key scientific applications and rationale include:

• Neuroscience and Synaptic Biology: Ephrin-B3 regulates excitatory synapse density and synaptic maturation, making it essential for dissecting mechanisms of synaptic plasticity, neural circuit formation, and neurodevelopmental disorders. It mediates cell-cell competition that determines synapse density, which is critical for understanding neural connectivity.

• Cancer Research: Ephrin-B3 is highly expressed in certain tumors, such as glioblastoma, where it acts as a survival factor by inhibiting apoptosis (cell death) and promoting angiogenesis (formation of new blood vessels). Recombinant Ephrin-B3 can be used to model these effects in vitro and in vivo, and to screen for potential therapeutic inhibitors targeting Eph/ephrin signaling.

• Immunology: Ephrin-B3 can induce the migration of memory B cells and modulate immune responses, making it useful for studying immune cell trafficking and the role of Eph/ephrin signaling in inflammation and immune regulation.

• Viral Pathogenesis: Ephrin-B3 functions as a cellular receptor for Nipah virus, so recombinant protein can be used in viral entry assays, receptor binding studies, and antiviral drug screening.

• Cell Signaling Studies: As a ligand for EphB receptors, recombinant Ephrin-B3 is used to activate or inhibit Eph receptor signaling in various cell types, enabling detailed analysis of downstream signaling pathways, receptor-ligand specificity, and bidirectional signaling mechanisms.

• Assay Development and Screening: Recombinant Ephrin-B3 is suitable for use in bioactivity assays, ELISA, Western blotting, and cell-based functional assays to study protein-protein interactions, receptor activation, and cellular responses.

• Standardization and Reproducibility: Using recombinant protein ensures batch-to-batch consistency, defined activity, and the absence of confounding factors present in tissue-derived preparations, which is critical for reproducible experimental results.

In summary, Recombinant Human Ephrin-B3 is a versatile tool for dissecting Eph/ephrin biology in neural, immune, and cancer systems, and for developing new therapeutic strategies targeting these pathways.

You can use recombinant human Ephrin-B3 as a standard for quantification or calibration in your ELISA assays, provided the protein is sufficiently purified and its concentration is accurately determined. This approach is common in quantitative ELISA workflows, where a standard curve is generated using known concentrations of the target protein.

Key considerations for using recombinant Ephrin-B3 as an ELISA standard:

• Purity and Characterization: The recombinant protein should be highly purified, ideally with minimal contaminants, and its concentration should be verified using an independent method such as HPLC or spectrophotometry.
• Formulation: Carrier proteins like BSA are sometimes added to recombinant proteins to enhance stability. For ELISA standards, both carrier-free and carrier-containing formulations can be used, but carrier-free is preferred if BSA may interfere with your assay.
• Calibration: Recombinant proteins are not inherently calibrated for ELISA use. You must calibrate them against a mass-calibrated standard or assign their value based on measurement in your specific ELISA system. Lot-to-lot variability and dilution errors can affect quantification, so always prepare standards carefully and validate your standard curve.
• Standard Curve Preparation: Prepare a dilution series covering the expected concentration range in your samples (commonly 0–1000 pg/mL, but adjust as needed). Follow best practices for reconstitution and dilution to minimize errors.
• Validation: Confirm that the recombinant Ephrin-B3 is recognized by your ELISA antibodies and produces a reliable, linear standard curve. This ensures accurate quantification of unknowns.

In summary, recombinant human Ephrin-B3 is suitable as an ELISA standard if you ensure proper purification, concentration determination, and calibration within your assay system. Always validate its performance in your specific ELISA setup.

Recombinant Human Ephrin-B3 has been validated for several applications in published research, primarily in studies of bioactivity, cell signaling, and functional assays related to neural and cancer biology.

Key validated applications include:

• Bioactivity assays: Recombinant Ephrin-B3 is routinely validated for its ability to engage Eph receptors and modulate downstream signaling pathways in cell-based assays.
• Neurite outgrowth inhibition: Used to demonstrate its role as a myelin-based inhibitor of neurite outgrowth in primary CNS neuron cultures, relevant to studies of axon guidance and spinal cord injury.
• Cell survival and apoptosis assays: Applied in glioblastoma research to show its function as a survival factor for tumor and endothelial cells, both in vitro (cell culture) and in vivo (zebrafish, CAM assay).
• Synapse density modulation: Utilized in neuronal co-culture systems to study its effect on excitatory synapse density via EphB signaling, with quantification by immunostaining for synaptic markers.
• Viral receptor studies: Used as a functional receptor in assays detecting henipavirus (Nipah and Hendra) entry, due to its high affinity for viral glycoproteins.
• Cell migration and axon guidance: Employed in studies of cortical interneuron migration and axon repulsion, often in co-culture or explant assays.
• Protein-protein interaction studies: Used in ELISA, Western blotting, and SDS-PAGE to confirm binding and molecular weight.

Additional details:

• In vitro and in vivo angiogenesis assays: Ephrin-B3 has been used in chorioallantoic membrane (CAM) assays and zebrafish models to study its role in promoting angiogenesis.
• Receptor-ligand binding studies: Applied in biochemical assays to characterize interactions with EphB and EphA receptors, including competitive binding and signaling activation.
• Standardization and controls: Used as a standard or positive control in ELISA and other immunoassays.

These applications are supported by both commercial validation and peer-reviewed research, particularly in the fields of neuroscience, oncology, and virology.

To reconstitute and prepare Recombinant Human Ephrin-B3 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) or sterile distilled water to achieve a final concentration typically between 0.1–1.0 mg/mL. For most bioactivity assays and cell culture applications, a concentration of ≥100 µg/mL is recommended.

Step-by-step protocol:

• Add the appropriate volume of sterile PBS or sterile distilled water directly to the vial containing the lyophilized protein. For example, to prepare a 1 mg/mL solution from 100 µg of protein, add 100 µL of buffer.
• Gently mix by swirling or inverting the vial. Do not vortex or pipette vigorously, as this can denature or aggregate the protein.
• Allow the protein to fully dissolve at room temperature for several minutes. If necessary, gently flick the tube to aid dissolution.
• Once reconstituted, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Store aliquots at –20°C for long-term storage or at 2–8°C for short-term use (up to one week).

Additional notes for cell culture experiments:

• If the protein is to be used for receptor activation, note that Fc-clustered or membrane-bound forms are typically required for full receptor activation; soluble monomeric forms may bind but not activate Eph receptors.
• For enhanced stability, some protocols recommend adding glycerol (5–50% final concentration) to the reconstituted solution, especially for storage or repeated use.
• Filter-sterilize the final solution if sterility is critical for your cell culture system.

Summary Table:

Precautions:

• Avoid vigorous mixing.
• Aliquot to prevent freeze-thaw cycles.
• Use sterile technique throughout.

These steps will ensure optimal solubility and bioactivity of Recombinant Human Ephrin-B3 protein for cell culture experiments.