Recombinant Human Ephrin-A4

Recombinant Human Ephrin-A4 is a valuable tool in research because it enables precise investigation of Eph/ephrin signaling, which is critical in neurobiology, cancer, immunology, and regenerative medicine. Its use allows for controlled, reproducible studies of ligand-receptor interactions, cellular responses, and therapeutic targeting.

Key reasons to use Recombinant Human Ephrin-A4 in research applications:

• Functional Studies of Eph Receptors: Ephrin-A4 is a high-affinity ligand for multiple Eph receptors (including EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, and EphB1), making it essential for dissecting receptor-specific signaling pathways in neural development, axon guidance, synaptic plasticity, and neurogenesis.
• Disease Modeling: Ephrin-A4/EphA4 signaling is implicated in neurodegenerative diseases (such as Alzheimer’s disease), depression, demyelination, and cancer progression. Recombinant Ephrin-A4 enables in vitro and in vivo modeling of these disease processes and the testing of potential inhibitors or therapeutic interventions.
• Cellular and Molecular Assays: Recombinant Ephrin-A4 is widely used in ELISA, Western blotting, inhibition assays, and cell-based functional assays to study receptor activation, downstream signaling, and cellular responses such as migration, adhesion, and differentiation.
• Therapeutic Target Validation: By providing a defined ligand, recombinant Ephrin-A4 allows for the screening and validation of small molecule inhibitors, antibodies, or peptides that modulate Eph/ephrin interactions, supporting drug discovery efforts for neurological and oncological indications.
• Immunological Research: Ephrin-A4 and its receptors play roles in immune cell development, activation, and migration, making recombinant forms useful for studying immune signaling and cell-cell communication.

Technical advantages of using recombinant proteins include:

• High purity and batch-to-batch consistency, ensuring reproducible results.
• Defined molecular structure (e.g., Fc chimeras or tagged versions) for specific assay requirements and enhanced stability.
• Suitability for a range of applications, including 3D cell culture, gene therapy research, and spatial biology studies.

In summary, Recombinant Human Ephrin-A4 is a critical reagent for elucidating Eph/ephrin biology, modeling disease, validating therapeutic targets, and performing a wide array of molecular and cellular assays in both basic and translational research contexts.

Recombinant Human Ephrin-A4 can be used as a standard for quantification or calibration in ELISA assays, but only if it is validated for this purpose within your specific assay system. The suitability depends on the compatibility of the recombinant protein with the antibodies used in your ELISA, its purity, and its formulation.

Key considerations:

• Validation Required: Not all ELISA kits are guaranteed to detect recombinant proteins equivalently to native proteins. Some manufacturers explicitly state that their kits may not reliably detect recombinant Ephrin-A4, and recommend using the standards provided with the kit, which are calibrated against a master calibrator.
• Formulation: Recombinant proteins formulated with stabilizers (e.g., BSA) are often recommended for use as ELISA standards, as they better mimic the conditions of the kit calibrators and improve stability.
• Immunoreactivity: The recombinant Ephrin-A4 must retain the epitopes recognized by both the capture and detection antibodies in your ELISA. Some recombinant preparations are validated for use in functional ELISAs, but this does not guarantee quantitative equivalence unless specifically tested.
• Assignment of Value: If you use recombinant Ephrin-A4 as a standard, it is best practice to assign its concentration based on measurement within the ELISA itself, rather than relying solely on the mass stated on the vial. This accounts for potential discrepancies in immunoreactivity and recovery.
• Recovery and Linearity: You should verify that the recombinant standard yields a linear response and acceptable recovery across the assay’s dynamic range. This typically involves spiking known amounts into sample matrix and comparing measured values to expected ones.

Best practices:

• Run a pilot experiment to compare the recombinant Ephrin-A4 standard curve to the kit’s native standard curve.
• Confirm that the recombinant protein produces a linear, reproducible standard curve within the assay’s validated range.
• Assign concentrations based on ELISA measurements, not just vial label mass, due to possible differences in immunologically recognizable mass.

Summary Table: Recombinant Standard Use in ELISA

In conclusion: You can use recombinant Human Ephrin-A4 as a standard for ELISA quantification if you validate its performance in your assay, ensure it is immunologically compatible, and assign its value based on ELISA results rather than vial mass.

Recombinant Human Ephrin-A4 has been validated for several applications in published research, primarily in the context of molecular and cellular biology assays. The most commonly reported validated applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or as a ligand to study Eph receptor binding and signaling.
• Western Blotting: Utilized as a positive control or to confirm protein expression and antibody specificity.
• Cell-based Assays: Applied in studies of cell signaling, receptor-ligand interactions, and functional assays such as growth cone collapse and axon guidance.
• 3D Cell Culture: Used to investigate cell-cell interactions and signaling in more physiologically relevant models.
• Inhibition Assays: Employed to block Eph receptor signaling, often as a soluble ligand or antagonist in functional studies.
• Spatial Biology and Mass Cytometry (CyTOF): Used in advanced multiplexed assays to study protein localization and cell phenotyping.
• Vaccine Development: Investigated as a potential immunogen or target in preclinical studies.

Supporting details and context:

• In in vitro and in vivo models, recombinant Ephrin-A4 has been used to probe Eph receptor function, particularly EphA4, in neurobiology, cancer, and regenerative medicine research.
• As a soluble ligand or Fc chimera, it is commonly used to antagonize Eph receptor signaling, enabling studies of axon guidance, synaptic plasticity, and disease models such as spinal cord injury and neurodegeneration.
• ELISA and Western blotting are standard validation methods for recombinant Ephrin-A4, confirming its activity and specificity in binding assays and protein detection.
• Inhibition assays with recombinant Ephrin-A4 have been used to dissect signaling pathways and validate therapeutic targets in both basic and translational research.

These applications are supported by multiple published studies and product validation notes, reflecting the broad utility of recombinant human Ephrin-A4 in molecular, cellular, and translational research contexts.

To reconstitute and prepare Recombinant Human Ephrin-A4 protein for cell culture experiments, first centrifuge the vial to collect the lyophilized powder at the bottom, then reconstitute it in sterile distilled water or sterile PBS to a final concentration of 0.1–0.5 mg/mL, depending on your experimental requirements. Avoid vigorous mixing to preserve protein integrity.

Detailed protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitution:
  • Add sterile distilled water or sterile PBS to achieve a concentration of 0.1–0.5 mg/mL (for example, add 200 μL to 100 μg of protein for 0.5 mg/mL).
  • If your application requires, you may use PBS (phosphate-buffered saline) instead of water, especially for Fc-chimeric forms or when the protein will be used in physiological conditions.
• Mix gently:
  • Do not vortex or pipette vigorously; gently swirl or invert the vial to dissolve the protein.
• Aliquot and storage:
  • Prepare small aliquots to avoid repeated freeze-thaw cycles, which can denature the protein.
  • Store reconstituted aliquots at –20 °C or –80 °C for long-term storage, or at 4 °C for short-term use (up to 2–7 days).
• Optional additives:
  • For enhanced stability, especially for long-term storage, consider adding 5–50% glycerol as a cryoprotectant.
  • If using in cell culture, ensure the final buffer is compatible with your cells (e.g., avoid high salt or non-physiological pH).

Best practices for cell culture:

• Use sterile technique throughout to prevent contamination.
• If the protein is to be added directly to cell culture media, dilute the reconstituted stock to the desired working concentration using sterile culture medium.
• Filter-sterilize the final working solution if sterility is in doubt.

Summary of key points:

• Centrifuge vial before opening.
• Reconstitute at 0.1–0.5 mg/mL in sterile water or PBS.
• Mix gently, avoid vigorous agitation.
• Aliquot and store at –20 °C or lower.
• Avoid repeated freeze-thaw cycles.

Always consult the specific product datasheet for any unique instructions related to your recombinant protein preparation.