Recombinant Mouse Ephrin-A2

Using Recombinant Mouse Ephrin-A2 in research applications is valuable for studying cell signaling, neural development, tissue morphogenesis, and disease mechanisms, particularly those involving the Eph/ephrin pathway. Ephrin-A2 is a membrane-bound ligand for Eph receptors, and recombinant forms enable controlled, reproducible activation or inhibition of these pathways in vitro and in vivo.

Key scientific applications and rationales include:

• Neural Development and Axon Guidance: Ephrin-A2 is critical for axon pathfinding, especially in the developing nervous system, where it provides repulsive cues to guide retinal ganglion cell axons and hippocampal axons to their correct targets. Recombinant ephrin-A2 can be used to mimic or block these signals in cell culture or animal models to dissect mechanisms of neural circuit formation.

• Neurogenesis and Cortical Organization: Ephrin-A2 regulates the balance between proliferation and differentiation of neural progenitors, influencing the formation and migration of excitatory and inhibitory neurons in the neocortex. Recombinant ephrin-A2 is used experimentally to activate forward or reverse signaling, allowing researchers to study its effects on neural stem cell behavior, cortical lamination, and potential links to neurodevelopmental disorders such as autism spectrum disorder.

• Cell Migration and Tissue Morphogenesis: Ephrin-A2/Eph receptor interactions are central to cell positioning, migration, and tissue boundary formation during embryogenesis and organ development. Recombinant ephrin-A2 enables in vitro assays (e.g., migration, adhesion, or morphogenesis assays) to elucidate these processes.

• Cancer and Disease Models: Ephrin-A2 is implicated in tumor biology, including cell migration, invasion, and the establishment of pre-metastatic niches. Recombinant protein can be used to probe these pathways in cancer cell lines or animal models.

• Functional Studies and Mechanistic Dissection: Recombinant ephrin-A2 allows for precise, dose-dependent activation or inhibition of Eph/ephrin signaling, facilitating mechanistic studies that are not possible with genetic models alone. It is commonly used in:

  • Ligand-receptor binding assays
  • Cell signaling pathway analysis
  • Functional blocking or activation experiments in cell culture or tissue explants

• Standardization and Reproducibility: Recombinant proteins provide a consistent, defined reagent for experiments, reducing variability compared to tissue extracts or conditioned media.

In summary, recombinant mouse ephrin-A2 is a powerful tool for dissecting the roles of Eph/ephrin signaling in development, disease, and cellular communication, enabling both basic research and translational studies.

Recombinant Mouse Ephrin-A2 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. This is a common practice in quantitative ELISA development, but several critical factors must be considered to ensure accuracy and reliability.

Key considerations and supporting details:

• Recombinant proteins are widely used as ELISA standards: Guidelines for ELISA standard curve preparation recommend using a purified protein, and if unavailable, a recombinant protein of known concentration is acceptable for generating a standard curve. This allows for precise quantification of the target analyte in unknown samples.

• Validation is essential: The recombinant Ephrin-A2 must be validated for use as a standard in your specific ELISA. This includes confirming that the recombinant protein is recognized by the capture and detection antibodies in your assay, and that its signal response is linear and comparable to the native protein in your sample matrix. Validation steps typically include:

  • Testing for parallelism between the standard curve (using recombinant protein) and serial dilutions of native samples.
  • Assessing spike-and-recovery and dilution linearity to ensure the recombinant standard behaves similarly to endogenous Ephrin-A2.
  • Confirming specificity to avoid cross-reactivity with related proteins.

• Recombinant Ephrin-A2 is commonly supplied for this purpose: Several sources explicitly state that recombinant mouse Ephrin-A2 (including Fc chimeras) is suitable for use as an ELISA standard, especially when provided with or without carrier proteins such as BSA. Carrier-free forms are preferred for standard curve preparation to avoid interference.

• Kit-specific recommendations: Some commercial ELISA kits are designed to detect only native (not recombinant) Ephrin-A2, so recombinant standards may not be appropriate for those kits unless validated. Always consult the kit documentation and, if necessary, perform your own validation experiments.

• Standard curve preparation: Prepare a dilution series of the recombinant protein in the same buffer or matrix as your samples to generate a standard curve, typically covering the expected range of analyte concentrations in your samples.

Summary Table: Use of Recombinant Mouse Ephrin-A2 as ELISA Standard

In conclusion: You can use recombinant mouse Ephrin-A2 as a standard for ELISA quantification, but you must validate its performance in your assay context to ensure accurate and reliable results.

Recombinant Mouse Ephrin-A2 has been validated in published research for several key applications, primarily in studies of neuronal development, cell signaling, and cancer biology.

Validated Applications in Published Research:

• Neuronal Differentiation and Migration Assays:
  Recombinant ephrin-A2 (often as an Fc chimera) has been used to activate ephrin-A2 forward and reverse signaling in in vitro and ex vivo assays to study its role in excitatory neuron differentiation, apical progenitor proliferation, and interneuron migration in the developing mouse neocortex. Researchers have applied clustered recombinant ephrin-A2-Fc to dissociated embryonic neocortex cultures and organotypic slice cultures to dissect signaling mechanisms.

• Cell Proliferation and Stem Cell Regulation:
  Addition of recombinant ephrin-A2 to neural progenitor cultures has been used to assess its effect on cell proliferation, demonstrating that ephrin-A2 reverse signaling can inhibit proliferation of adult stem cells in the mouse subventricular zone.

• Cancer Cell Migration and Invasion Assays:
  Recombinant ephrin-A2 Fc chimeras have been used to stimulate glioma cell lines in in vitro migration and invasion assays, showing that ephrin-A2 promotes glioblastoma cell invasion, likely through modulation of EphA2 phosphorylation and downstream signaling pathways.

• Binding Activity and Receptor-Ligand Interaction Studies:
  Recombinant mouse ephrin-A2 has been validated in binding activity assays to study interactions with Eph receptors, particularly EphA2, which is relevant for understanding cell signaling in both normal physiology and disease contexts such as cancer and viral entry.

• Functional Blocking and Neutralization:
  Recombinant ephrin-A2 has been used in functional assays to block or neutralize Eph receptor signaling, helping to dissect the specific roles of ephrin-A2/EphA2 interactions in various cellular processes.

Additional Context:

• ELISA and Western Blot:
  While not always using the recombinant protein directly as an analyte, antibodies against mouse ephrin-A2 have been validated for use in ELISA and Western blot, often with recombinant ephrin-A2 as a positive control or standard.

• Disease Models:
  Recombinant ephrin-A2 has been used in models of neurodevelopmental disorders (e.g., autism spectrum disorder) and cancer to elucidate its functional roles in vivo and in vitro.

Summary Table of Applications

These applications demonstrate that recombinant mouse ephrin-A2 is a versatile tool for dissecting Eph/ephrin signaling in neurobiology, oncology, and cell biology research.

To reconstitute and prepare Recombinant Mouse Ephrin-A2 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) at a concentration of 250 μg/mL. This concentration is commonly recommended for Ephrin-A2 Fc chimera proteins and provides a suitable starting point for most cell-based assays.

Essential steps and best practices:

• Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
• Add sterile PBS directly to the vial to achieve the desired concentration (e.g., 250 μg/mL).
• Gently mix by pipetting up and down or by slow vortexing; avoid vigorous agitation to prevent protein denaturation.
• Allow the protein to fully dissolve at room temperature for several minutes. Inspect visually to confirm complete dissolution.
• Aliquot the solution if you do not plan to use the entire volume immediately. This minimizes freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at −20°C or −80°C in a manual defrost freezer. Avoid repeated freeze-thaw cycles.
• For short-term use, the reconstituted protein can be stored at 4°C for up to one week.

Preparation for cell culture experiments:

• Dilute the reconstituted stock to the working concentration required for your assay using sterile PBS or cell culture medium. Typical working concentrations range from 0.1–10 μg/mL, but optimal conditions should be determined empirically for each cell type and experimental setup.
• If using the protein to stimulate cells, consider clustering the Ephrin-A2 protein (e.g., by pre-incubation with anti-Fc antibodies if using an Fc chimera) to mimic membrane-bound ligand activity, as soluble monomeric forms may not fully activate Eph receptors.
• Always use aseptic technique to prevent contamination.

Additional notes:

• Consult the specific product datasheet for any unique requirements regarding buffer composition, pH, or additives (e.g., carrier proteins like BSA) to enhance stability.
• If the protein is not an Fc chimera, confirm the recommended buffer and concentration from the manufacturer or literature, as some recombinant proteins may require Tris-HCl or other buffers for optimal solubility.

This protocol ensures the recombinant Ephrin-A2 protein is properly reconstituted and ready for use in cell culture experiments, maintaining its biological activity and stability.