Recombinant Mouse EphA4

Recombinant mouse EphA4 serves as a valuable research tool for investigating the Eph-ephrin signaling system, which plays critical roles across multiple biological processes and disease contexts.

Key Research Applications

Neurological Disease Modeling

Recombinant mouse EphA4 is particularly valuable for studying Alzheimer's disease pathology. The protein enables investigation of how EphA4 mediates amyloid-beta-induced synaptic dysfunction and impaired long-term potentiation. By using recombinant EphA4 in experimental systems, researchers can evaluate therapeutic interventions targeting the EphA4 ligand-binding domain and assess whether blocking EphA4 signaling rescues synaptic deficits in disease models. This application extends to understanding EphA4's role in other neurological conditions, including spinal cord injury recovery and locomotor coordination defects.

Osteoarthritis Research

The recombinant protein facilitates investigation of EphA4's dual regulatory properties in joint tissue. EphA4 signaling demonstrates anti-catabolic effects on osteoclasts and synoviocytes while exhibiting pro-anabolic effects on articular chondrocytes. Recombinant EphA4 ligands (such as EfnA4-fc chimeric proteins) can be administered to evaluate therapeutic strategies for both preventing and treating osteoarthritis in preclinical models.

Drug Discovery and Development

Recombinant mouse EphA4 serves as an essential reagent for high-throughput screening and structure-activity relationship studies. The protein enables identification of novel EphA4 inhibitors through virtual screening approaches and validation of candidate compounds in biochemical assays. This application accelerates the discovery of both small-molecule inhibitors and repurposed FDA-approved drugs targeting EphA4.

Mechanistic Studies

The recombinant protein allows researchers to examine Eph-ephrin interactions involved in cell migration, tissue morphogenesis, and cellular signaling cascades. These fundamental studies provide insights into the molecular mechanisms underlying EphA4's physiological and pathological roles across multiple biological systems.

Yes, you can use recombinant Mouse EphA4 as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant proteins are commonly used as standards in ELISA protocols, especially when purified native protein is unavailable.

Essential context and supporting details:

• Recognition by ELISA: Many commercial Mouse EphA4 ELISA kits specify that their standard curve is generated using recombinant Mouse EphA4, and the assay is validated to recognize both natural and recombinant forms of the protein. This ensures that the recombinant protein is suitable for calibration, as long as the antibodies used in your ELISA recognize the recombinant form equivalently to the native protein.

• Purity and Activity: The recombinant Mouse EphA4 you referenced is highly purified (>98% by SDS-PAGE), with low endotoxin levels and confirmed bioactivity (e.g., binding to ephrin ligands). These characteristics are important for reliable standard curve generation.

• Standard Curve Preparation: When preparing ELISA standards, it is critical to use a well-characterized, purified protein. Recombinant proteins are preferred when native proteins are not available, and their concentration should be accurately determined (e.g., by absorbance at 280 nm or BCA assay). Serial dilutions of the recombinant protein are used to generate the standard curve, which allows quantification of unknown samples by interpolation.

• Assay Validation: Before routine use, confirm that your ELISA antibodies do not discriminate between recombinant and native EphA4. Most kits report no significant cross-reactivity or interference, but this should be verified for your specific assay. Some kits are designed to detect only native EphA4, so always check the kit documentation.

• Formulation Considerations: Recombinant proteins may be supplied in buffers containing stabilizers (e.g., trehalose, mannitol, Tween 80). Ensure these do not interfere with your ELISA reagents or detection system.

Best practices:

• Validate the recombinant EphA4 standard in your assay by comparing its response to that of native EphA4, if available.
• Prepare standards using the same buffer as your samples to minimize matrix effects.
• Store and handle the recombinant protein according to manufacturer recommendations to preserve activity and stability.

Limitations:

• If your ELISA kit or antibodies are specific for native conformations or post-translational modifications not present in the recombinant protein, quantification may be inaccurate.
• Always consult the ELISA kit manual and perform pilot experiments to confirm suitability.

Summary Table: Recombinant Mouse EphA4 as ELISA Standard

In conclusion: Recombinant Mouse EphA4 is suitable as a standard for ELISA quantification if validated for your assay, and is widely used for this purpose in research settings.

Recombinant Mouse EphA4 has been validated for several key applications in published research, primarily in studies of neurodegenerative diseases, synaptic function, and joint pathology.

Validated Applications:

• In vitro binding assays: Recombinant Mouse EphA4 ectodomain has been immobilized for ligand-binding studies, such as ELISA-based assays to screen for inhibitors or characterize ligand interactions.
• Cellular signaling studies: Used to investigate EphA4-dependent signaling pathways in cultured neurons, including growth cone collapse assays and synaptic transmission studies.
• Animal model interventions: Applied in mouse models to study the effects of EphA4 blockade or activation on disease phenotypes, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and osteoarthritis (OA).
• Therapeutic candidate validation: Utilized to test the efficacy of small-molecule inhibitors or agonists targeting EphA4, assessing their ability to rescue synaptic plasticity or prevent cell death in disease models.
• Protein-protein interaction studies: Used to demonstrate direct interactions with other receptor tyrosine kinases, such as PDGFRβ, in co-expression systems.

Supporting Details:

• In AD research, recombinant EphA4 was used to show that blocking its ligand-binding domain reverses synaptic impairment and rescues long-term potentiation deficits in mouse models.
• In ALS models, EphA4 agonists prevented astrocyte-mediated motor neuron toxicity in vitro, demonstrating its utility in neuroprotection assays.
• In OA studies, EphA4-Fc fusion proteins were injected into injured joints to modulate disease progression, highlighting its application in in vivo therapeutic validation.
• EphA4 has also been used in protein interaction assays to elucidate its role in signaling networks relevant to neural development and disease.

Summary Table of Applications

These applications demonstrate the versatility of recombinant Mouse EphA4 in mechanistic studies, drug discovery, and therapeutic validation across multiple disease models.

To reconstitute and prepare Recombinant Mouse EphA4 protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water or sterile phosphate-buffered saline (PBS), typically to a final concentration between 0.1–1.0 mg/mL. The exact buffer and concentration should be confirmed by consulting the product-specific datasheet or certificate of analysis, as some formulations may require specific conditions.

Step-by-step protocol:

• Centrifuge the vial briefly to ensure all lyophilized powder is at the bottom before opening.
• Add sterile water or PBS: For most recombinant proteins, sterile distilled water or PBS is suitable. For example, add 100–1000 μL to 100 μg of protein to achieve 1 mg/mL to 0.1 mg/mL.
• Gently mix: Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation. Avoid vigorous shaking or vortexing to prevent foaming and protein denaturation.
• Optional additives: For enhanced stability, especially during storage, you may add carrier proteins (e.g., 0.1% BSA) or glycerol (5–50% final concentration) if compatible with your downstream application.
• Aliquot and store: Prepare small aliquots to avoid repeated freeze-thaw cycles. Store reconstituted protein at –20°C to –80°C for long-term storage. Avoid multiple freeze-thaw cycles, as these can denature the protein.
• Endotoxin testing: If using for sensitive cell culture applications, confirm low endotoxin levels (<0.01 EU/μg is typical for cell culture use).

Additional notes:

• Always consult the specific product datasheet for recommended buffer, concentration, and storage conditions, as requirements may vary depending on the protein tag, formulation, and intended application.
• If the protein is supplied in solution (not lyophilized), it may already be in PBS with glycerol and can be used directly or diluted as needed.
• For functional assays, confirm activity by binding to ephrin ligands or via ELISA as described in product literature.

Summary of best practices:

• Use sterile technique throughout.
• Avoid repeated freeze-thaw cycles.
• Confirm buffer compatibility with your cell culture system.
• Validate protein activity and purity before use in critical experiments.

These steps will help ensure the Recombinant Mouse EphA4 protein is properly prepared for cell culture applications.