Recombinant Mouse EphA2

Recombinant Mouse EphA2 is widely used in research due to its critical roles in cell signaling, development, disease modeling, and therapeutic target validation. Its applications span cell biology, cancer research, stem cell biology, and immunology.

Key scientific reasons to use Recombinant Mouse EphA2:

• Modeling Eph/ephrin signaling: EphA2 is a receptor tyrosine kinase that binds ephrin ligands, mediating cell-cell communication essential for tissue patterning, angiogenesis, and neural development. Recombinant EphA2 enables controlled studies of receptor-ligand interactions, kinase activation, and downstream signaling pathways in vitro.

• Cancer research and metastasis studies: EphA2 is implicated in tumor progression, metastasis, and vascular permeability. Recombinant EphA2 is used to investigate mechanisms of tumor cell migration, invasion, and the disruption of barriers such as the blood-brain barrier. It also serves as a target for screening inhibitors or antibodies that block its oncogenic activity.

• Stem cell biology and safety: EphA2 is a marker for undifferentiated pluripotent stem cells and is co-expressed with OCT4. Recombinant EphA2 can be used to develop assays for distinguishing undifferentiated (potentially tumorigenic) cells from differentiated populations, improving the safety of stem cell transplantation.

• Cell adhesion and migration assays: The extracellular region of EphA2 promotes cell adhesion and spreading, making recombinant protein valuable for studying cell-matrix and cell-cell interactions, especially in immune and endothelial cells.

• Bioassays and binding studies: Recombinant EphA2 is essential for quantitative binding assays, such as measuring ephrin ligand affinity, receptor clustering, and activation kinetics. It is also used in enzyme kinetics, inhibitor screening, and selectivity profiling.

• Preclinical therapeutic validation: Recombinant EphA2 is used to test monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates targeting EphA2, supporting drug development for cancer and other diseases.

Best practices for use:

• Optimize protein concentration and assay conditions for each application, as binding and activation depend on receptor clustering and ligand presentation.
• Use recombinant EphA2 in formats suitable for your assay (e.g., Fc chimera for clustering, His-tag for purification or ELISA).
• Confirm specificity using appropriate controls, as EphA2 shares high homology with other Eph receptors.

Summary of applications:

• Cell signaling and kinase activation assays
• Cancer cell migration, invasion, and metastasis models
• Stem cell sorting and safety assays
• Cell adhesion and spreading studies
• Therapeutic antibody and inhibitor screening
• Bioassays for ligand-receptor binding

Using recombinant Mouse EphA2 provides a controlled, reproducible tool for dissecting its biological functions and validating therapeutic strategies in diverse research fields.

Recombinant Mouse EphA2 protein can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This approach is widely accepted in ELISA development and quantification protocols.

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 purified protein is unavailable. Ensure the recombinant EphA2 is well-characterized, with its concentration measured by reliable methods such as absorbance at 280 nm or HPLC.

• Validation in ELISA: Published protocols and commercial kits often use recombinant EphA2 as a standard for quantification in ELISA assays. For example, a validated in-house ELISA for EphA2 used recombinant EphA2 protein to generate standard curves, confirming specificity, linearity, and recovery. Commercial recombinant mouse EphA2 proteins are also tested for ELISA applications.

• Formulation: If available, use a carrier-free or BSA-containing formulation depending on your assay needs. Carrier-free is preferred for direct quantification, while BSA-containing formulations may be more stable for repeated freeze-thaw cycles.

• Assay Optimization: Optimal dilutions and standard curve ranges should be empirically determined for your specific ELISA setup. The linear range for EphA2 detection in functional ELISA is typically in the low ng/mL range, but this must be validated for your assay.

• Species Specificity: Ensure the recombinant EphA2 matches the species of your target analyte (mouse EphA2 for mouse samples) to avoid cross-reactivity or calibration errors.

• Documentation: Record the lot number, concentration, and storage conditions of your recombinant standard for reproducibility and quality control.

Summary Table: Use of Recombinant Mouse EphA2 as ELISA Standard

In conclusion: You can use recombinant mouse EphA2 as a standard for ELISA quantification, provided you validate its performance in your specific assay and ensure its purity and concentration are well-characterized.

Recombinant Mouse EphA2 has been validated for several key applications in published research, primarily in bioassays, binding assays, and in vivo functional studies.

Validated Applications:

• Bioassay: Used to study receptor-ligand interactions, such as binding to ephrin-A1 and other ephrin ligands, and to assess downstream signaling events like kinase activation. For example, it has been employed to investigate macrophage adhesion, blood-brain barrier disruption in cerebral malaria, and cancer cell proliferation.

• Binding Assay / Protein-Protein Interaction: Utilized in solid-phase binding assays to quantify interactions with ligands (e.g., ephrin-A1, progranulin) and to characterize binding kinetics (e.g., affinity measurements via SPR). These assays are essential for validating EphA2 as a functional receptor and for screening potential inhibitors or therapeutic agents.

• In Vivo Studies: Applied in animal models to study EphA2’s role in physiological and pathological processes, such as vascular permeability in lung injury, tumor growth inhibition, and stem cell pluripotency regulation. Recombinant EphA2 has been used to probe its function in disease models and therapeutic interventions.

• Cell Surface Marker Validation: EphA2 has been validated as a marker for undifferentiated mouse embryonic stem cells (ESCs) using antibody-based cell sorting and flow cytometry, supporting its use in stem cell research and regenerative medicine.

Additional Context:

• Theranostics and Imaging: Recombinant Mouse EphA2 has been used to validate high-affinity peptide ligands for PET imaging and radiotheranostic applications, confirming its utility in preclinical cancer imaging and targeted therapy development.

• Enzyme Kinetics and Inhibitor Screening: The protein is suitable for enzyme kinetics studies, inhibitor screening, and selectivity profiling, supporting drug discovery efforts targeting EphA2.

• ELISA and Western Blot: While not as frequently cited for recombinant protein, antibodies against EphA2 are validated for ELISA and Western blot detection of endogenous or recombinant EphA2 in various sample types.

Summary Table:

Key Insights:

• Recombinant Mouse EphA2 is a versatile tool validated for mechanistic studies, therapeutic screening, and biomarker research.
• Its applications span basic receptor biology, disease modeling, drug discovery, and advanced imaging.

If you require detailed protocols or specific experimental setups for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Mouse EphA2 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) to a final concentration of 100 μg/mL.

Step-by-step protocol:

• Equilibrate the vial and PBS to room temperature before opening.
• Centrifuge the vial briefly to collect the powder at the bottom.
• Add sterile PBS to achieve the desired concentration (typically 100 μg/mL for EphA2).
• Gently mix by pipetting or gentle agitation for 15–30 minutes at room temperature until fully dissolved. If particulates remain, continue mixing for up to 2 hours.
• Aliquot the solution to avoid repeated freeze-thaw cycles.
• Storage:
  • For short-term use (up to 1 month), store at 2–8°C.
  • For long-term storage, keep at –20°C to –80°C in a manual defrost freezer.
• Avoid repeated freeze-thaw cycles to maintain protein activity.

Additional considerations for cell culture:

• Endotoxin levels: Confirm that the protein is endotoxin-tested and suitable for cell culture applications (typically <0.1 ng/μg).
• Sterility: Use sterile technique throughout to prevent contamination.
• Dilution for experiments: Prepare working solutions in sterile PBS or cell culture medium immediately before use. Optimal concentrations should be determined empirically for your specific assay.
• Carrier protein: If required for stability, add a carrier protein (e.g., 0.1% BSA) to working aliquots.

Summary Table:

Always consult the specific product datasheet for any additional instructions or variations in formulation.