Ephrin-A3, also known as EFNA3 encodes a member of the ephrin (EPH) family. The ephrins and EPH-related receptors comprise the largest subfamily of receptor protein-tyrosine kinases and have been implicated in mediating developmental events, especially in the nervous system and in erythropoiesis. 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. Ephrin-A3 encodes an EFNA class ephrin.
Protein Details
Purity
>90% by SDS-PAGE and analyzed by silver stain.
Endotoxin Level
<0.1 EU/µg as determined by the LAL method
Biological Activity
The biological activity of Human Ephrin-A3 was determined by its ability to compete with biotinylated human Ephrin-A3/Fc (BT359) for binding to immobilized mouse Eph-A6/Fc in a functional ELISA assay.
The predicted molecular weight of Recombinant Human Ephrin-A3 is Mr 47.7 kDa. However, the actual molecular weight as observed by migration on SDS-PAGE is Mr 60-70 kDa.
Predicted Molecular Mass
47.7
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.
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Recombinant Human Ephrin-A3 is a valuable tool in research due to its central role in cell signaling, particularly in neural development, cell migration, immune modulation, and cancer biology. Using the recombinant form allows for controlled, reproducible studies of Ephrin-A3’s biological functions and its interactions with Eph receptors.
Key reasons to use Recombinant Human Ephrin-A3 in research applications:
Modeling Eph/ephrin signaling: Ephrin-A3 is a ligand for Eph receptors, mediating contact-dependent, bidirectional signaling that regulates cell positioning, migration, and adhesion in both normal development and disease contexts. Recombinant protein enables precise in vitro studies of these pathways.
Neuroscience research: Ephrin-A3 is critical in axon guidance, synaptic plasticity, and neural stem cell regulation. It modulates pathways such as Wnt/β-catenin and influences neurogenesis, glial scar formation, and glutamate homeostasis, making it essential for studies of CNS development, injury, and repair.
Cancer and metastasis studies: Ephrin-A3 and its receptors are implicated in tumor progression, angiogenesis, and metastasis. Manipulating Ephrin-A3 signaling can alter tumor cell proliferation, migration, and the tumor microenvironment, providing a platform for therapeutic target validation and drug screening.
Immunology and cell biology: Ephrin-A3 is involved in immune cell modulation and tissue patterning. Recombinant protein is widely used in immunological assays, cell adhesion/migration studies, and protein-protein interaction analyses.
Therapeutic development: Because Eph/ephrin pathways are drug targets in cancer, neurodegeneration, and tissue regeneration, recombinant Ephrin-A3 is used in screening for inhibitors, antibodies, or other modulators.
Assay standardization: Recombinant proteins provide consistent quality and defined activity, essential for reproducible results in ELISA, binding assays, and functional studies.
Protein engineering and structural studies: Recombinant Ephrin-A3 is suitable for crystallography, biophysical characterization, and mapping interaction interfaces with Eph receptors.
In summary, Recombinant Human Ephrin-A3 is indispensable for dissecting Eph/ephrin signaling mechanisms, modeling disease processes, and developing targeted therapies in neuroscience, oncology, and immunology.
Yes, you can use Recombinant Human Ephrin-A3 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is purified and its concentration can be accurately measured.
Key Points for Using Recombinant Human Ephrin-A3 as an ELISA Standard:
Purified Protein: The recombinant protein should be highly purified. Many suppliers provide purified recombinant Ephrin-A3 (e.g., R&D Systems, Novus Biologicals, Leinco Technologies), which is suitable for use as an ELISA standard.
Accurate Concentration Measurement: The concentration of the recombinant protein should be determined using a reliable method such as HPLC, amino acid analysis, or spectrophotometry (A280). This is essential for generating a reliable standard curve.
Carrier Protein Consideration: Some recombinant proteins are supplied with a carrier protein (e.g., BSA). If the carrier protein does not interfere with your ELISA, it is generally acceptable to use the protein with BSA. However, if the carrier protein could interfere (e.g., in certain sandwich ELISAs), use a carrier-free version.
Standard Curve Preparation: Prepare a serial dilution series of the recombinant Ephrin-A3 to generate a standard curve that spans the expected concentration range of your samples. This will allow you to accurately quantify the amount of Ephrin-A3 in your unknown samples.
Validation: It is good practice to validate the standard by comparing it with a commercially available ELISA kit standard, if possible, to ensure consistency and accuracy.
Example Suppliers of Recombinant Human Ephrin-A3 Suitable for ELISA Standards:
R&D Systems: Recombinant Human Ephrin-A3 Fc Chimera Protein (with or without BSA)
Novus Biologicals: Recombinant Human Ephrin-A3 Protein
In summary, as long as the recombinant Human Ephrin-A3 is purified and its concentration is accurately known, it can be used effectively as a standard for quantification in ELISA assays.
Recombinant Human Ephrin-A3 has been validated in published research primarily for applications in cell signaling studies, neural stem cell proliferation assays, and receptor binding/activation experiments.
Key validated applications include:
Neural stem cell proliferation and differentiation assays: Recombinant ephrin-A3 has been used to study its role in suppressing Wnt signaling and inhibiting the proliferation of retinal stem cells (RSCs) both in vitro and in vivo. In these studies, antibody-clustered recombinant ephrin-A3 was applied to neural stem cell cultures to activate Eph receptor signaling, resulting in measurable changes in BrdU incorporation (a marker of cell proliferation) and neural differentiation markers.
Receptor binding and activation assays: Recombinant ephrin-A3 is commonly used to activate EphA receptors (such as EphA4) in cell-based assays, often by clustering the protein with antibodies to mimic physiological ligand-receptor interactions. This enables the study of downstream signaling pathways and functional outcomes.
Functional studies in cancer biology: Ephrin-A3 has been investigated for its role as a tumor suppressor in certain cancer cell types, such as malignant peripheral nerve sheath tumors, where recombinant protein is used to probe its effects on cell signaling and tumorigenic properties.
Binding competition and affinity assays: The recombinant protein is used in competitive binding assays to characterize interactions with Eph receptors, often employing biotinylated ephrin-A3 and immobilized receptor proteins.
Therapeutic development and screening: Recombinant ephrin-A3 serves as a tool in preclinical studies for therapeutic development, including screening for molecules that modulate Eph/ephrin signaling.
Summary Table of Validated Applications
Application Type
Description/Assay Details
Neural stem cell proliferation/differentiation
BrdU incorporation, neural marker expression, in vitro and in vivo stem cell assays
Receptor binding/activation
Antibody-clustered ephrin-A3 to activate EphA receptors, signaling pathway analysis
Cancer biology functional studies
Tumor suppressor role, signaling modulation in cancer cell lines
Binding competition/affinity assays
Competitive binding with biotinylated ephrin-A3, receptor-ligand interaction studies
Therapeutic development/screening
Functional studies for drug discovery and validation
Additional Notes:
Most published research uses recombinant ephrin-A3 in cell culture systems, often as an Fc chimera or tagged protein to facilitate clustering and receptor activation.
The protein is also used as a standard in ELISA and other immunoassays, though this is less frequently highlighted in primary research publications.
If you require details on a specific application or protocol, please specify the context (e.g., neural, cancer, immunoassay).
To reconstitute and prepare Recombinant Human Ephrin-A3 protein for cell culture experiments, first centrifuge the vial briefly to collect the lyophilized powder at the bottom. Then, reconstitute the protein in sterile distilled water or sterile PBS to a final concentration typically between 0.1–0.5 mg/mL (some protocols allow up to 1.0 mg/mL).
Step-by-step protocol:
Centrifuge the vial before opening to ensure all material is at the bottom.
Add sterile distilled water or PBS to achieve the desired concentration (commonly 0.1–0.5 mg/mL; for example, add 100–500 μL per 50 μg protein for 0.1–0.5 mg/mL).
Gently mix by pipetting up and down or swirling; avoid vigorous vortexing to prevent protein denaturation.
If the protein is difficult to dissolve, allow it to sit on ice for 10–30 minutes, then mix gently again.
Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
Store aliquots at −20 °C to −80 °C for long-term storage, or at 2–8 °C for up to one month.
Additional recommendations:
If the working concentration is below 0.5 mg/mL, consider adding a carrier protein (e.g., 0.1% BSA or HSA) to stabilize the protein during storage and handling.
Always consult the product-specific datasheet or certificate of analysis for any unique requirements, as some formulations may differ.
For cell culture, ensure the final buffer is compatible with your assay and does not contain toxic preservatives.
Summary Table:
Step
Details
Centrifuge vial
Briefly, before opening
Reconstitution
Sterile water or PBS, 0.1–0.5 mg/mL (up to 1.0 mg/mL)
Mixing
Gentle pipetting or swirling
Aliquoting
To minimize freeze-thaw cycles
Storage
−20 °C to −80 °C (long-term); 2–8 °C (short-term)
Carrier protein
Optional, 0.1% BSA/HSA if <0.5 mg/mL
These steps will ensure optimal solubility and biological activity of recombinant Ephrin-A3 for cell culture applications.
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
