Recombinant Mouse EphA3

Using Recombinant Mouse EphA3 in research applications is valuable for studying cell signaling, cancer biology, immunotherapy development, and neurobiology, due to its role as a receptor tyrosine kinase involved in cell adhesion, migration, and tumor progression.

Key scientific applications and rationale include:

• Cancer Research and Therapeutic Targeting: EphA3 is overexpressed in various tumors, including glioblastoma and bladder cancer, where it can serve as a tumor-specific marker and therapeutic target. Recombinant EphA3 protein is used to generate monoclonal antibodies, develop chimeric antigen receptor (CAR) T cells, and test antibody-drug conjugates in preclinical models. For example, immunizing mice with recombinant EphA3 enabled the production of high-affinity, specific antibodies for CAR T-cell therapy, which showed potent anti-tumor effects in glioblastoma models.

• Biomarker Discovery and Mechanistic Studies: EphA3 expression correlates with prognosis and tumor grade in cancers such as bladder cancer, where high EphA3 levels are associated with better outcomes and reduced tumor growth. Recombinant EphA3 is essential for in vitro assays (e.g., ELISA, Western blot, SPR) to quantify and characterize EphA3 interactions, signaling, and downstream effects.

• Cell Adhesion and Migration Studies: EphA3 regulates cytoskeletal dynamics and cell motility by modulating Rho and Ras family GTPases, impacting processes like cell migration, invasion, and tissue patterning. Recombinant protein allows for controlled stimulation or inhibition of EphA3 signaling in cultured cells, enabling mechanistic dissection of these pathways.

• Neuroscience and Regeneration: EphA3 is upregulated in astrocytes after nervous tissue injury and is involved in neural development and repair. Recombinant EphA3 can be used to study neuron-glia interactions, axon guidance, and regenerative responses in vitro and in vivo.

• Immunological Applications: EphA3 influences immune cell infiltration and the tumor microenvironment, making it relevant for immunotherapy research and immune profiling. Recombinant protein is used to screen for immune modulators or to develop immunotherapeutic agents.

• Tool for Antibody and Ligand Screening: Recombinant EphA3 serves as a standard antigen for screening and characterizing antibodies, small molecules, or peptides that bind or modulate EphA3 activity, facilitating drug discovery and validation workflows.

In summary, Recombinant Mouse EphA3 is a versatile reagent for dissecting EphA3 function, validating therapeutic strategies, and modeling disease processes in both basic and translational research contexts.

You can use recombinant mouse EphA3 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately known.

Key considerations and supporting details:

• Purity and Quantification: For ELISA calibration, the standard should be a purified protein with a well-defined concentration. Recombinant mouse EphA3 proteins are commonly used as standards in commercial ELISA kits and are validated for this purpose. Ensure your recombinant EphA3 is at least 90% pure (as determined by SDS-PAGE or similar methods) and that its concentration has been reliably measured, typically by absorbance at 280 nm or BCA assay.

• Form and Tag: The recombinant EphA3 should ideally match the form detected by your assay (e.g., extracellular domain, tag type). Fc- or His-tagged versions are frequently used as standards, but the tag should not interfere with antibody recognition in your ELISA.

• Standard Curve Preparation: Reconstitute the lyophilized recombinant protein according to the manufacturer’s instructions, then prepare a serial dilution to generate a standard curve covering the expected range of your samples. The standard curve should bracket the concentrations you expect in your experimental samples.

• Validation: Confirm that your ELISA antibodies recognize the recombinant EphA3 standard equivalently to the native protein in your samples. This is especially important if your samples are complex matrices (e.g., serum, tissue lysate), as matrix effects can influence quantification.

• Conjugation: If your recombinant EphA3 is conjugated to a fluorophore (e.g., Alexa Fluor 555), ensure this does not interfere with antibody binding or detection in your ELISA format. For colorimetric or chemiluminescent ELISAs, unconjugated or appropriately tagged proteins are preferred.

• Documentation: Many commercial ELISA kits for mouse EphA3 use recombinant EphA3 as the standard, and protocols specify reconstitution and dilution steps for standard curve generation.

Summary Table: Considerations for Using Recombinant Mouse EphA3 as an ELISA Standard

Conclusion:
If your recombinant mouse EphA3 meets these criteria, it is suitable as a standard for ELISA quantification and calibration. Always validate the standard curve and assay performance with your specific reagents and sample types.

Recombinant Mouse EphA3 has been validated for several applications in published research, primarily in the following contexts:

• Immunization and Antibody Generation: Recombinant mouse EphA3 protein has been used as an immunogen to generate EphA3-specific monoclonal antibodies in mice. These antibodies were then characterized for specificity and affinity using assays such as ELISA and surface plasmon resonance.

• Chimeric Antigen Receptor (CAR) T Cell Therapy Development: The recombinant protein was used to develop and validate EphA3-targeted CAR T cells. These CAR T cells were tested for their ability to recognize and kill EphA3-expressing glioblastoma cells in vitro and in vivo, including in patient-derived glioblastoma neurospheres, organoids, and xenograft mouse models.

• Western Blotting: Recombinant mouse EphA3 has been validated as a positive control or antigen in Western blot assays to confirm antibody specificity and to detect EphA3 expression in various tissues or cell lines.

• ELISA (Enzyme-Linked Immunosorbent Assay): The protein is used as a standard or coating antigen in direct ELISA assays to assess antibody binding and specificity.

• Bioactivity Assays: Recombinant EphA3 has been used in bioactivity assays to study its functional effects, such as its role in cell signaling, migration, and interaction with other proteins.

• In Vivo Functional Studies: The protein has been used to study EphA3’s biological roles, such as its involvement in tumor growth, immune cell infiltration, and tissue remodeling, often through overexpression or silencing in mouse models.

Summary Table of Validated Applications

Key details:

• In glioblastoma research, recombinant mouse EphA3 was central to the development and validation of both monoclonal antibodies and CAR T cell therapies, with extensive in vitro and in vivo validation.
• In cancer and fibrosis models, EphA3 protein was used to study its biological function and therapeutic potential, including effects on tumor growth and immune cell regulation.
• For antibody validation, recombinant EphA3 is routinely used in ELISA and Western blot protocols to confirm specificity and cross-reactivity.

If you require protocols or more technical details for a specific application, please specify the intended use.

To reconstitute and prepare Recombinant Mouse EphA3 protein for cell culture experiments, follow these general guidelines based on standard protocols and product information:

Reconstitution

1. Centrifugation: Before opening the vial, centrifuge it briefly to ensure all lyophilized powder is at the bottom.
2. Solvent: Reconstitute the lyophilized protein in sterile, deionized water or sterile PBS (phosphate-buffered saline), depending on the manufacturer’s instructions. For most Mouse EphA3 proteins, sterile water is recommended.
3. Concentration: Reconstitute to a concentration between 0.1–1.0 mg/mL. Some sources recommend a minimum concentration of 0.1 mg/mL for optimal stability and activity.
4. Gentle Mixing: Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing, as this can denature the protein or cause foaming.
5. Incubation: Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete reconstitution.

Preparation for Cell Culture

1. Aliquoting: After reconstitution, aliquot the protein solution into small volumes to minimize freeze-thaw cycles, which can reduce protein activity.
2. Storage: Store aliquots at –20°C or lower. For long-term storage, some protocols recommend adding 5–50% glycerol (final concentration) to prevent freezing and maintain stability.
3. Sterility: Ensure all steps are performed under sterile conditions to avoid contamination, especially if the protein will be used directly in cell culture.
4. Carrier Protein: For extended storage or to enhance stability, consider adding a carrier protein such as 0.1% BSA (bovine serum albumin) or 5% trehalose.

Example Protocol

• Centrifuge the vial before opening.
• Add sterile deionized water to achieve a concentration of 0.1–1.0 mg/mL.
• Gently mix and allow to dissolve at room temperature for 15–30 minutes.
• Aliquot and store at –20°C. For long-term storage, add 5–50% glycerol or 0.1% BSA.
• Thaw aliquots on ice before use and avoid repeated freeze-thaw cycles.

Notes

• Always refer to the specific product datasheet for any unique requirements or recommendations.
• Ensure the protein is fully dissolved before use in cell culture experiments.
• Maintain sterility throughout the process to prevent contamination of cell cultures.

These steps will help ensure the recombinant Mouse EphA3 protein is properly reconstituted and prepared for use in cell culture experiments.