Recombinant Mouse EphA6

Using Recombinant Mouse EphA6 in research applications is valuable for studying the molecular mechanisms of Eph receptor signaling, particularly in cancer biology, neurobiology, and angiogenesis. EphA6 is a member of the Eph receptor tyrosine kinase family, which plays critical roles in cell adhesion, migration, axon guidance, and tumor progression.

Key scientific applications and rationales include:

• Cancer Research: EphA6 has been implicated in both tumor suppression and promotion, depending on the context. In glioblastoma, increased EphA6 expression sensitizes glioma-initiating cells to BMP-2-induced apoptosis, suggesting a tumor-suppressive role and potential as a therapeutic target or biomarker. Conversely, in prostate cancer, EphA6 promotes angiogenesis, invasion, and metastasis, making it a candidate for studying mechanisms of tumor progression and for developing anti-metastatic therapies.

• Developmental and Cell Biology: Eph receptors, including EphA6, are essential for developmental processes such as axon guidance, synapse formation, and tissue patterning. Recombinant EphA6 can be used to dissect these pathways in vitro, for example, by studying its effects on neuronal or endothelial cell behavior.

• Angiogenesis Studies: EphA6 contributes to neovascularization in tumors. Recombinant EphA6 can be used in endothelial cell assays to investigate its role in tube formation and vascular remodeling, which are relevant for both cancer and developmental biology.

• Protein-Protein Interaction and Signaling Pathways: Recombinant EphA6 enables controlled studies of receptor-ligand interactions, downstream signaling events, and the formation of receptor complexes, such as with BMP type I receptors in apoptosis pathways.

• Therapeutic Target Validation: Given its dual roles in tumor biology, recombinant EphA6 is useful for screening inhibitors, antibodies, or other modulators that may have therapeutic potential in cancer or other diseases involving Eph/ephrin signaling.

• Biochemical and Structural Studies: Recombinant proteins allow for detailed biochemical characterization, including phosphorylation, glycosylation, and binding assays, as well as structural studies to elucidate receptor domains and conformational changes.

In summary, recombinant Mouse EphA6 is a versatile tool for elucidating the functional roles of EphA6 in health and disease, validating therapeutic strategies, and advancing our understanding of Eph receptor biology in both normal and pathological contexts.

You can use recombinant Mouse EphA6 protein as a standard for quantification or calibration in ELISA assays, provided the protein is highly purified, its concentration is accurately determined, and it is compatible with your assay's detection antibodies and format.

For ELISA quantification, the standard must:

• Be of high purity (typically >95% by SDS-PAGE).
• Have a known and verified concentration (e.g., determined by absorbance at 280 nm or BCA assay).
• Be biologically active and recognized by the antibodies used in your ELISA.
• Be reconstituted and diluted according to best practices to maintain stability and reproducibility.

Key considerations:

• The recombinant Mouse EphA6 described is >97% pure, expressed in HEK293 cells, and supplied lyophilized in PBS with protectants. It is measured for bioactivity in a functional ELISA, indicating suitability for binding-based assays.
• For quantitative ELISA, you must prepare a standard curve using serial dilutions of the recombinant protein, covering the expected range of your samples.
• The standard should be run in parallel with your samples in each assay to ensure accurate quantification.
• Confirm that your ELISA antibodies specifically recognize the recombinant EphA6 and do not cross-react with other proteins.
• If using a carrier-free or BSA-containing formulation, select the one appropriate for your assay format and avoid matrix effects.

Best practices for ELISA standards:

• Use freshly reconstituted aliquots and avoid repeated freeze-thaw cycles.
• Validate the linearity and reproducibility of your standard curve in preliminary experiments.
• Ensure the recombinant protein's epitope matches the native protein in your samples for accurate quantification.

In summary, recombinant Mouse EphA6 can serve as a standard for ELISA quantification if it meets purity, concentration, and antibody recognition requirements, and is properly validated in your assay system.

Recombinant Mouse EphA6 has been validated primarily for binding assays (functional ELISA) and SDS-PAGE, with its biological activity confirmed by its ability to bind ephrin ligands in vitro. Published research and product datasheets indicate the following validated applications:

• Functional ELISA (Binding Assays): Recombinant mouse EphA6 has been shown to bind recombinant human EphrinA3 in a dose-dependent manner, confirming its biological activity and suitability for ligand-receptor interaction studies.
• SDS-PAGE: The protein has been validated for purity and molecular weight assessment using SDS-PAGE.
• Western Blot (WB): While direct evidence for the recombinant protein itself in WB is limited, antibodies raised against recombinant EphA6 fragments are validated for Western blot detection of EphA6, suggesting the recombinant protein can serve as a positive control or immunogen in this application.
• ELISA (Direct): The recombinant protein is used as a standard or antigen in direct ELISA assays for antibody validation and quantification.
• Immunogen for Antibody Production: Recombinant fragments of EphA6 have been used as immunogens to generate polyclonal and monoclonal antibodies, which are then validated in various immunoassays.

Additional potential applications (based on recombinant protein best practices and supplier protocols, though not always explicitly validated in peer-reviewed publications for this specific protein) include:

• Immunoprecipitation (IP): Some suppliers list IP as a potential application for recombinant EphA6.
• Flow Cytometry (FC): Occasionally listed as a possible application for recombinant proteins, though direct validation for EphA6 is not clearly documented in the search results.

Summary Table: Validated Applications for Recombinant Mouse EphA6

Key context:

• The most robust published validation is for binding assays (functional ELISA), confirming the protein’s biological activity.
• SDS-PAGE is routinely used for quality control and is validated for this protein.
• Use as an immunogen is well established for antibody generation.
• Applications such as immunoprecipitation and flow cytometry are suggested by supplier protocols but lack direct peer-reviewed validation in the search results.

If you require validation for a specific application not listed here, it is advisable to consult primary literature or perform in-house validation using established protocols.

To reconstitute and prepare Recombinant Mouse EphA6 protein for cell culture experiments, add sterile water to the lyophilized protein to achieve the recommended stock concentration, typically 0.2–0.25 mg/mL, and gently mix to dissolve without vigorous agitation. Centrifuge the vial briefly at 4°C before opening to recover all contents.

Step-by-step protocol:

• Preparation:

  • Confirm the protein is lyophilized from sterile PBS, pH 7.4, often with protectants such as trehalose, mannitol, and Tween-80.
  • Review the Certificate of Analysis (CoA) for specific buffer details and recommended concentration.

• Reconstitution:

  • Add sterile, endotoxin-free water directly to the vial to reach the desired stock concentration (e.g., 0.2 mg/mL or 0.25 mg/mL).
  • Gently agitate or invert the vial for 15–30 minutes at room temperature to fully dissolve the protein. Avoid vigorous shaking or vortexing to prevent foaming and protein denaturation.
  • If necessary, measure concentration by UV-Vis spectrophotometry.

• Aliquoting and Storage:

  • Once dissolved, aliquot the solution into sterile tubes to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C to –80°C for long-term use. For short-term use (2–7 days), store at 4–8°C.
  • Avoid repeated freeze-thaw cycles to maintain protein integrity.

• Preparation for Cell Culture:

  • Before adding to cell culture, dilute the stock solution into sterile cell culture medium or buffer compatible with your assay. Ensure the final buffer conditions (e.g., PBS, pH 7.4) are suitable for your cells and do not contain toxic additives.
  • If required, add carrier proteins (e.g., BSA) to stabilize the recombinant protein during dilution and application.
  • Filter-sterilize the working solution if sterility is critical for your experiment.

Additional notes:

• Confirm the endotoxin level is <1.0 EU/μg, which is suitable for most cell culture applications.
• Always consult the product datasheet or CoA for any special instructions or buffer components that may affect cell viability.
• For functional assays, typical working concentrations range from 0.31–10 ng/mL for binding studies, but optimal concentrations should be empirically determined for your specific cell culture system.

This protocol ensures the recombinant EphA6 protein is properly reconstituted, aliquoted, and prepared for reliable use in cell culture experiments.