Recombinant Mouse Ephrin-A4

Recombinant Mouse Ephrin-A4 is a valuable tool for research applications focused on neurobiology, immunology, and cell signaling due to its role as a ligand for Eph receptors, which regulate processes such as axon guidance, synaptic plasticity, myelination, immune cell interactions, and tissue morphogenesis.

Key scientific reasons to use recombinant Mouse Ephrin-A4 include:

• Modeling Eph-Ephrin Signaling: Ephrin-A4 is a membrane-bound ligand for EphA receptors, especially EphA4, which are critical in neural development, axon guidance, and synaptic function. Recombinant Ephrin-A4 enables controlled studies of receptor-ligand interactions in vitro, facilitating mechanistic investigations of signaling pathways.

• Neurobiology and Disease Research: Ephrin-A4/EphA4 signaling has been implicated in demyelination, depression, and neurodegenerative diseases. Recombinant Ephrin-A4 can be used to probe these pathways, screen for inhibitors, or model disease states in cell-based assays and animal models.

• Functional Assays: Recombinant Ephrin-A4 is suitable for use in cell culture, ELISA, and biochemical assays to study cell adhesion, migration, and morphology. It can serve as a ligand to activate or block Eph receptor signaling, allowing for precise manipulation of cellular responses.

• Immunology Applications: Ephrin-A4 may play a role in immune cell development and interactions, such as between B lymphocytes and dendritic cells. Recombinant protein can be used to dissect these processes in vitro.

• Standardization and Reproducibility: Using recombinant protein ensures batch-to-batch consistency, defined purity, and concentration, which are critical for quantitative assays and reproducible experimental results.

Typical applications include:

• Cell signaling studies (e.g., receptor activation, downstream pathway analysis)
• Neural regeneration and repair models
• Drug screening for EphA4 inhibitors in neurodegenerative and psychiatric disease contexts
• Cell adhesion and migration assays
• ELISA standards and controls for antibody validation

In summary, recombinant Mouse Ephrin-A4 is essential for dissecting Eph-ephrin biology, modeling disease mechanisms, and developing therapeutic strategies targeting EphA4 signaling pathways.

You can use recombinant Mouse Ephrin-A4 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 recognized by the antibodies used in your assay.

Key considerations and supporting details:

• Purity and Quantification: For ELISA calibration, the standard should be a purified protein with a known concentration. Recombinant proteins are commonly used for this purpose, especially when native protein is unavailable or difficult to purify. Ensure the recombinant Ephrin-A4 is well-characterized and quantified, ideally using methods such as HPLC or BCA assay.

• Antibody Recognition: The recombinant protein must be recognized by the capture and detection antibodies in your ELISA. Some ELISA kits are validated to detect both recombinant and native Ephrin-A4, while others are specific for the native form and may not recognize recombinant standards due to differences in folding, post-translational modifications, or tags. Always check the kit documentation or perform a pilot experiment to confirm compatibility.

• Standard Curve Preparation: Prepare the standard curve using serial dilutions of the recombinant Ephrin-A4 in the same buffer or matrix as your samples to minimize matrix effects. Follow the ELISA kit’s instructions for standard preparation, as some kits provide specific diluents or protocols for optimal results.

• Kit-Specific Recommendations: Some commercial ELISA kits for mouse Ephrin-A4 explicitly state that they recognize both recombinant and native proteins, making recombinant standards suitable for quantification. However, other kits are designed to detect only the native form and may not be suitable for use with recombinant standards. Always consult the kit manual or technical support for guidance.

Summary Table: Recombinant Protein as ELISA Standard

If your recombinant Mouse Ephrin-A4 meets these criteria and is recognized by your ELISA antibodies, it is appropriate to use it as a standard for quantification or calibration in your assays. If uncertain, perform a validation experiment to compare the response of the recombinant standard to that of native protein, if available.

Recombinant Mouse Ephrin-A4 has been validated for several key applications in published research, primarily in the fields of neurobiology, immunology, and vascular biology.

Validated Applications:

• Cell-based assays: Recombinant Mouse Ephrin-A4 is widely used in in vitro assays to study Eph/ephrin signaling, including receptor-ligand binding, activation, and downstream signaling events in neuronal and endothelial cells.
• ELISA (Enzyme-Linked Immunosorbent Assay): It serves as a standard or capture protein for quantifying Ephrin-A4 or its interactions in biological samples.
• Western blotting: Recombinant Ephrin-A4 is used as a positive control or antigen for antibody validation in Western blot assays.
• Functional blocking and decoy experiments: The Fc-chimera form of recombinant Ephrin-A4 is used to inhibit Eph receptor signaling by acting as a decoy ligand, particularly in studies of vascular leak, sepsis, and neurodegenerative disease models.
• Neuronal growth and guidance assays: It is employed to investigate axon guidance, synaptic plasticity, and neurogenesis, often by modulating EphA4 signaling in cultured neurons or animal models.
• Immunological studies: Recombinant Ephrin-A4 is used to study interactions between B-lymphocytes and dendritic cells, especially in tonsillar tissue.
• Tissue culture: It is applied in cell and tissue culture systems to probe Eph/ephrin-mediated cellular responses, including migration, adhesion, and differentiation.

Supporting Details:

• In sepsis models, Ephrin-A4-Fc was used to inhibit Eph/ephrin signaling, resulting in reduced vascular leak and improved survival in mice.
• In neurobiology, recombinant Ephrin-A4 has been used to study its role in demyelination, depression, and synaptic deficits, often through knockdown or blocking experiments.
• ELISA validation is supported by its use as a standard or antigen in direct binding assays.
• Western blot validation is indicated by its use as a control antigen for antibody specificity testing.
• Functional studies often utilize the Fc-chimera format to block endogenous Eph receptor signaling in both in vitro and in vivo systems.

Additional Notes:

• Recombinant Mouse Ephrin-A4 is typically expressed in mammalian cells (e.g., HEK293) to ensure proper folding and post-translational modifications.
• It is available in carrier-free and BSA-containing formulations, with carrier-free versions preferred for ELISA and cell culture applications to avoid interference.
• The protein is used in research on cancer cell biology, cell and gene therapy, and CNS disorders, reflecting its broad utility in both basic and translational studies.

Summary Table:

These applications are supported by peer-reviewed studies and product documentation, confirming the utility of recombinant Mouse Ephrin-A4 in diverse experimental contexts.

To reconstitute and prepare Recombinant Mouse Ephrin-A4 protein for cell culture experiments, dissolve the lyophilized protein at 200 μg/mL in sterile PBS. This concentration is widely recommended for Ephrin-A4 Fc chimera preparations.

Step-by-step protocol:

• Equilibrate the vial and PBS to room temperature before opening.
• Centrifuge the vial briefly to collect the lyophilized powder at the bottom.
• Add sterile PBS to achieve a final concentration of 200 μg/mL. For example, add 500 μL PBS to 100 μg protein, or 1 mL PBS to 200 μg protein.
• Gently mix by pipetting up and down or by gentle inversion. Avoid vigorous vortexing, which may denature the protein.
• Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation. If undissolved flakes remain, continue mixing for up to 2 hours.
• Aliquot the reconstituted solution to avoid repeated freeze-thaw cycles.
• Storage after reconstitution:
  • Store at 2–8 °C for up to 1 month under sterile conditions.
  • For longer-term storage, keep aliquots at –20 °C to –70 °C for up to 3 months.
• Avoid repeated freeze-thaw cycles to maintain protein integrity.

Additional notes for cell culture use:

• If using for receptor activation, note that Fc-clustered or membrane-bound ephrin ligands are required for robust receptor activation; soluble monomeric forms may bind but not activate Eph receptors.
• For cell culture experiments, dilute the reconstituted stock to the desired working concentration in sterile culture medium or PBS immediately before use.
• If required for stability or low concentration applications, consider adding a carrier protein (e.g., 0.1% BSA) to prevent adsorption to plasticware.

Summary Table:

This protocol ensures optimal solubility and stability for Recombinant Mouse Ephrin-A4 protein in cell culture experiments.