Ephrin-A5, also known as EFNA5, is a member of the ephrin gene family that prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. The EPH and EPH-related receptors comprise the largest subfamily of receptor protein-tyrosine kinases and have been implicated in mediating developmental events, particularly in the nervous system. EPH receptors typically have a single kinase domain and an extracellular region containing a Cys-rich domain and 2 fibronectin type III repeats. Based on their structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. The Eph family of receptors are similarly divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. Ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.1
The predicted molecular weight of Recombinant Human Ephrin-A5 is Mr 48.6 kDa. However, the actual molecular weight as observed by migration on SDS-PAGE is Mr 50-55 kDa.
Predicted Molecular Mass
48.6
Formulation
This recombinant protein was 0.2 µm filtered and lyophilized from modified Dulbecco’s phosphate buffered saline (1X PBS) pH 7.2 – 7.3 with no calcium, magnesium, or preservatives.
Storage and Stability
This lyophilized protein is stable for six to twelve months when stored desiccated at -20°C to -70°C. After aseptic reconstitution, this protein may be stored at 2°C to 8°C for one month or at -20°C to -70°C in a manual defrost freezer. Avoid Repeated Freeze Thaw Cycles. See Product Insert for exact lot specific storage instructions.
Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.
Recombinant Human Ephrin-A5 is widely used in research applications to study cell signaling, neural development, cancer biology, and cell migration due to its role as a ligand for Eph receptor tyrosine kinases, which mediate critical cell-cell communication events.
Key scientific reasons to use Recombinant Human Ephrin-A5:
Neural Development and Guidance: Ephrin-A5 is essential for axon guidance, growth cone collapse, and neurite retraction, making it a valuable tool for investigating mechanisms of neuronal migration, patterning, and synaptic connectivity in developmental neurobiology. It can exert both repulsive and attractive effects on migrating neurons, depending on context.
Cell Signaling Studies: Ephrin-A5 activates EphA receptors, triggering downstream phosphorylation events and modulating signaling networks involved in cell adhesion, motility, and aggregation. Recombinant forms allow precise control in bioassays and binding studies to dissect these pathways.
Cancer Research: Ephrin-A5 functions as a tumor suppressor and is implicated in the regulation of cell proliferation, migration, and invasion in various cancers, including glioma and ovarian cancer. Recombinant Ephrin-A5 can be used to study its effects on cancer cell behavior and as a potential biomarker for aggressive tumor phenotypes.
Functional Assays: Recombinant Ephrin-A5 is validated for use in bioassays, immunoprecipitation, coprecipitation, immunocytochemistry (ICC), and immunohistochemistry (IHC), enabling diverse experimental approaches to analyze protein-protein interactions, receptor activation, and cellular responses.
Mechanistic Studies: It is used to investigate mechanisms such as receptor internalization, trafficking, and co-adaptation in cell signaling, particularly in the context of Eph/ephrin interactions.
Best practices for use:
Pre-cluster recombinant Ephrin-A5 with secondary antibodies to mimic physiological ligand presentation and enhance receptor activation in cell-based assays.
Employ in dose-response bioassays to quantify effects on cell motility, aggregation, or neurite outgrowth.
Use in combination with EphA receptor-expressing cells or tissues to study specific ligand-receptor interactions and downstream signaling events.
Recombinant Human Ephrin-A5 provides a reproducible, well-characterized tool for dissecting Eph/ephrin signaling pathways and their biological consequences in both basic and translational research contexts.
Recombinant Human Ephrin-A5 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly formulated and validated for this purpose.
For use as an ELISA standard:
Formulation matters: Recombinant proteins with added Bovine Serum Albumin (BSA) are generally recommended for use as ELISA standards because BSA enhances protein stability and shelf-life. Carrier-free formulations (without BSA) are suitable if BSA could interfere with your assay, but may be less stable.
Validation: The recombinant Ephrin-A5 should be validated for quantitative use in your specific ELISA format. This includes confirming its purity, concentration, and biological activity. Many commercial ELISA kits use recombinant Ephrin-A5 as a standard for calibration curves.
Calibration curve: Prepare a serial dilution of the recombinant Ephrin-A5 to generate a standard curve, which allows quantification of unknown samples by comparison to known concentrations.
Compatibility: Ensure the recombinant protein matches the native form detected by your assay antibodies (e.g., Fc fusion, tag-free, glycosylation status), as differences may affect antibody recognition and quantification accuracy.
Best practices:
Use the same buffer and diluent conditions for standards and samples to minimize matrix effects.
Store and handle the recombinant protein according to manufacturer recommendations to preserve activity and stability.
Validate the linearity, sensitivity, and reproducibility of your standard curve in pilot experiments before routine use.
In summary, recombinant Human Ephrin-A5 is suitable as an ELISA standard if it is appropriately formulated and validated for your assay conditions. Always confirm compatibility with your specific ELISA antibodies and protocol.
Recombinant Human Ephrin-A5 has been validated for a range of applications in published research, primarily in studies of cell signaling, neural development, cancer biology, and cell migration. The most commonly reported applications include:
Bioassays: Used to study Eph/ephrin signaling, neural progenitor proliferation, neuron survival, tumor suppression, and cell migration in various cell types and tissues.
Immunoprecipitation: Applied to investigate protein-protein interactions, such as Ephrin-A5 acting as a ligand for Eph receptors in neural and cancer cell contexts.
Coprecipitation Assays: Used to analyze direct binding and complex formation between Ephrin-A5 and its receptor partners.
Immunohistochemistry (IHC): Utilized to localize Ephrin-A5 expression in tissue sections, including studies of neuronal compartments and tumor tissues.
In Vivo Studies: Employed in animal models to assess the physiological and pathological roles of Ephrin-A5, such as in neural development and hormone regulation.
Cellular Adaptation and Trafficking Assays: Used to study the dynamics of Ephrin-A5 on the cell surface and its intracellular trafficking, particularly in neuronal growth cones.
Binding Activity Assays: Validated for measuring the binding of Ephrin-A5 to Eph receptors, often using Fc-chimeric forms for enhanced detection.
Antiproliferative and Cytotoxicity Assays: Used to test the effects of Ephrin-A5 on cancer cell proliferation and survival, including the development of Ephrin-A5-based cytotoxins for targeted cancer therapy.
Western Blot and ELISA: Some sources recommend Ephrin-A5 for use in Western blotting and ELISA, though these are less frequently cited in primary research articles.
Cell Motility and Migration Assays: Ephrin-A5 has been used to modulate and study cell motility, particularly in cancer and neural cells.
Representative research contexts:
Neural development: Ephrin-A5 is widely used to study axon guidance, neuronal differentiation, and synaptic organization.
Cancer biology: Validated for investigating tumor suppressor functions, receptor signaling in glioma and ovarian cancer, and as a targeting moiety for cytotoxins.
Stem cell differentiation: Used in protocols converting pluripotent stem cells into neuronal subtypes.
These applications are supported by peer-reviewed studies and product validation data, demonstrating the versatility of recombinant human Ephrin-A5 in both basic and translational research.
To reconstitute and prepare Recombinant Human Ephrin-A5 protein for cell culture experiments, follow these steps:
Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
Reconstitution buffer: Most protocols recommend using sterile PBS (phosphate-buffered saline) or sterile distilled water. For Fc-tagged or chimera forms, sterile PBS is preferred. For untagged or non-chimera forms, sterile distilled water is often suitable.
Concentration: Reconstitute to a final concentration of 0.1–0.5 mg/mL in sterile distilled water or ≥100–200 μg/mL in sterile PBS for Fc-tagged proteins.
Dissolving: Gently swirl or invert the vial to dissolve. Do not vortex or pipette vigorously to avoid protein denaturation.
Aliquoting: Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
Storage:
Short-term: Store reconstituted protein at 2–8°C for up to 1 week.
Long-term: Store aliquots at –20°C or –80°C for up to several months. Avoid repeated freeze-thaw cycles.
Preparation for cell culture:
Before adding to cell culture, dilute the reconstituted stock to the desired working concentration using cell culture medium or buffer compatible with your assay.
If using in sensitive cell types, consider adding a carrier protein (e.g., 0.1% BSA) to minimize adsorption and stabilize the protein.
Summary Table:
Step
Recommendation
Notes
Centrifuge vial
Yes
Before opening
Buffer
Sterile PBS (Fc-tagged) or sterile water (untagged)
Check datasheet for your product
Concentration
0.1–0.5 mg/mL (water) or ≥100–200 μg/mL (PBS)
Adjust as needed for your application
Mixing
Gentle swirling/inversion
No vortexing or harsh pipetting
Aliquot
Yes
To avoid freeze-thaw cycles
Storage
2–8°C (short), –20°C/–80°C (long)
Follow product-specific guidance
Additional notes:
Always consult the specific product datasheet for any unique instructions, as formulations and tags may vary.
For functional assays, confirm the biological activity post-reconstitution if possible.
These steps will help ensure the stability and activity of recombinant Ephrin-A5 for your cell culture experiments.
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
