Recombinant Mouse Ephrin-A1

Recombinant Mouse Ephrin-A1 is widely used in research because it is a key ligand for Eph receptors, particularly EphA2, and plays critical roles in cell signaling, migration, tissue morphogenesis, angiogenesis, and cancer progression. Using the recombinant form allows for controlled, reproducible studies of these biological processes in vitro and in vivo.

Key scientific applications and rationales include:

• Studying Eph-Ephrin signaling: Ephrin-A1 is essential for investigating the mechanisms of Eph receptor activation, which regulates cell positioning, boundary formation, and tissue patterning during development and in adult tissues.
• Cancer research: Ephrin-A1 is implicated in tumor growth, metastasis, and neovascularization. It can induce epithelial-mesenchymal transition (EMT), promote cancer cell migration, and facilitate metastasis by activating both EphA2 and EGFR signaling pathways. Recombinant Ephrin-A1 is used to model these processes and to screen for inhibitors or therapeutic interventions.
• Angiogenesis and vascular biology: Ephrin-A1 and its receptors are crucial for blood vessel formation and remodeling, making the recombinant protein valuable for angiogenesis assays and studies of tumor vasculature.
• Cell migration and adhesion assays: Recombinant Ephrin-A1 is used to stimulate or inhibit cell migration, adhesion, and spreading in various cell types, providing insights into developmental biology and disease mechanisms.
• Biochemical and structural studies: The recombinant protein is suitable for binding assays, immunoprecipitation, ELISA, and structural analyses to dissect ligand-receptor interactions and downstream signaling events.
• Biomarker and therapeutic target research: Soluble Ephrin-A1 levels are elevated in certain cancers and disease states, making it a potential biomarker and therapeutic target.

Using recombinant protein ensures batch-to-batch consistency, defined activity, and the ability to manipulate concentration and presentation (e.g., Fc-chimeras for clustering or biotinylated forms for immobilization), which are critical for reproducible experimental outcomes.

In summary, Recombinant Mouse Ephrin-A1 is a versatile tool for dissecting Eph/Ephrin biology, modeling disease processes, and developing targeted therapies in cancer, vascular biology, and developmental studies.

Yes, recombinant Mouse Ephrin-A1 can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately known. This is a common practice in quantitative ELISA, where a standard curve is generated using known concentrations of a purified or recombinant protein to enable precise quantification of the target analyte in samples.

Key considerations and best practices:

• Purity and Formulation: The recombinant protein should be highly purified. Carrier proteins such as BSA are often added to enhance stability and shelf-life, and formulations with BSA are generally recommended for use as ELISA standards. Carrier-free versions are available if BSA interferes with your assay, but these may be less stable and require careful handling.
• Concentration Determination: The concentration of the recombinant Ephrin-A1 must be accurately determined, typically by absorbance at 280 nm or another validated method, to ensure the standard curve is reliable.
• Standard Curve Preparation: Prepare a dilution series covering the expected range of your samples (commonly 0–1000 pg/mL, but this can be adjusted as needed). Follow the reconstitution and dilution instructions specific to your protein lot for best results.
• Validation: Ensure that the recombinant standard is recognized by the antibodies used in your ELISA. Some kits are validated only for native protein, not recombinant forms, so check your kit documentation. If using a custom or in-house ELISA, confirm that the recombinant protein is detected equivalently to the native form.
• Documentation: Some ELISA kits provide their own standards, which are validated for that specific assay. If using a different recombinant standard, you may need to validate its performance in your assay system.

Limitations and caveats:

• Epitope Recognition: Recombinant proteins may differ from native proteins in post-translational modifications or folding, potentially affecting antibody recognition. This is especially relevant if your ELISA antibodies are conformation-dependent.
• Kit-Specific Recommendations: Some commercial ELISA kits specify that their standards are for native protein only and may not be compatible with recombinant standards. Always consult the kit datasheet.
• Bioassay vs. ELISA Standard: Recombinant proteins labeled as "ELISA standard" are not always validated for use in functional bioassays and vice versa.

Summary Table: Recombinant Mouse Ephrin-A1 as ELISA Standard

In summary: Recombinant Mouse Ephrin-A1 is suitable as a standard for ELISA quantification if it is pure, accurately quantified, and recognized by your assay antibodies. Always validate its performance in your specific assay context.

Recombinant Mouse Ephrin-A1 has been validated for several key applications in published research, primarily in bioassays, immunoprecipitation, and as a binding activity standard. The most common use is as a functional ligand to study Eph receptor signaling, cell migration, tissue morphogenesis, cancer progression, and vascular biology.

Validated Applications in Published Research:

• Bioassays:
  Recombinant Mouse Ephrin-A1 is widely used in cell-based bioassays to activate Eph receptors (such as EphA2) and investigate downstream signaling pathways. These assays have been applied to study:

  • Regulation of cell migration and tissue morphogenesis.
  • Cancer cell signaling, including epithelial-mesenchymal transition (EMT) and metastasis.
  • Endothelial cell function and vascular integrity, especially in models of sepsis and cerebral malaria.
  • Tumor neovascularization and angiogenesis.
  • Schwann cell migration and neural cell interactions.
  • Species-specific receptor binding in virology.

• Immunoprecipitation:
  Used to pull down Ephrin-A1 or its interacting Eph receptors from cell lysates, enabling analysis of protein-protein interactions and post-translational modifications.

• Binding Activity:
  Validated as a standard for receptor-ligand binding assays, including ELISA and surface plasmon resonance, to quantify Ephrin-A1/Eph receptor interactions.

• Functional Ligand Studies:
  Ephrin-A1-Fc fusion proteins are used to mimic membrane-bound ephrin-A1, allowing researchers to probe receptor activation, phosphorylation, and internalization in various cell types.

• In Vivo Models:
  Recombinant Ephrin-A1 has been used in mouse models to study its role in cancer metastasis, vascular leak, and therapeutic interventions targeting Eph/ephrin signaling.

Additional Context:

• Sample Types:
  Most studies use whole cells or cell lysates from mouse, human, or other mammalian species.

• Research Areas:
  Applications span cancer biology, neurobiology, vascular biology, immunology, and developmental biology.

• Protocols:
  Recombinant Mouse Ephrin-A1 is typically used in soluble form (often as an Fc chimera) for cell stimulation, receptor binding, or as a positive control in signaling assays.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant mouse Ephrin-A1 in diverse experimental settings.

To reconstitute and prepare Recombinant Mouse Ephrin-A1 protein for cell culture experiments, follow these steps:

• Reconstitution:
  Add sterile phosphate-buffered saline (PBS) to the lyophilized protein to achieve a final concentration of 100 μg/mL. Briefly centrifuge the vial before opening to ensure all material is at the bottom. Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.

• Buffer Choice:
  Use sterile PBS (pH 7.2–7.4) unless otherwise specified in your datasheet. Some protocols allow reconstitution in sterile distilled water, but PBS is preferred for stability and physiological compatibility.

• Aliquoting and Storage:
  After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.

  • Store aliquots at –80°C for long-term storage (up to 12 months).
  • For short-term use (up to 1 month), store at 2–8°C.
  • Avoid repeated freeze-thaw cycles.

• Preparation for Cell Culture:

  • If using a carrier-free formulation, consider adding a small amount of carrier protein (e.g., 0.1% BSA) if stability is a concern, unless BSA interferes with your assay.
  • Filter-sterilize the reconstituted protein if sterility is critical and your protocol allows.
  • Dilute the reconstituted stock to the desired working concentration in cell culture medium immediately before use. Typical working concentrations for functional assays range from 1–10 μg/mL, but optimal concentrations should be determined empirically for your specific application.

• General Best Practices:

  • Always handle recombinant proteins using sterile technique.
  • If the protein is fused to Fc or other tags, confirm compatibility with your downstream assay.
  • Consult the specific product datasheet for any unique instructions regarding buffer composition, stabilizers, or additives.

Summary Table: Recombinant Mouse Ephrin-A1 Reconstitution

References:

• Lyophilized from 0.2 μm filtered PBS, reconstitute at 100 μg/mL in sterile PBS. Avoid repeated freeze-thaw cycles.
• Reconstitution in PBS or sterile water, gentle mixing, aliquoting, and storage recommendations.
• Storage and stability guidelines.
• General recombinant protein handling.

If your experiment requires a specific buffer or additive (e.g., for enhanced stability or activity), refer to the product datasheet or published protocols for further optimization.