Recombinant Human EphA1

Recombinant Human EphA1 is a valuable tool for research applications due to its central role in cell signaling pathways that regulate critical biological processes such as cell adhesion, migration, angiogenesis, proliferation, and apoptosis. Here are several reasons why you should consider using Recombinant Human EphA1 in your research:

1. Study of Eph/ephrin Signaling Pathways: EphA1 is a member of the Eph receptor family, which interacts with ephrin ligands to mediate bidirectional signaling. Using recombinant EphA1 allows you to investigate the molecular mechanisms of Eph/ephrin signaling, including downstream events such as activation of kinases, modulation of cell adhesion, and regulation of cell motility.

2. Cancer Research: EphA1 is implicated in tumor invasiveness and progression in various carcinomas. Recombinant EphA1 can be used to study its role in tumor cell behavior, including migration, invasion, and angiogenesis, and to evaluate its potential as a prognostic marker or therapeutic target in cancer.

3. Angiogenesis and Vascular Biology: EphA1, in conjunction with ephrin-A1, plays a role in endothelial cell function and angiogenesis. Recombinant EphA1 can help elucidate the mechanisms underlying vascular development and remodeling, which are relevant to both physiological and pathological conditions.

4. Immune Cell Regulation: Eph receptors and ephrins are involved in immune cell development, activation, and migration. Recombinant EphA1 can be used to explore its effects on immune cell adhesion, retention at inflammatory sites, and interactions with other immune signaling pathways.

5. Neurological and Developmental Studies: Eph receptors are crucial in nervous system development and function. Recombinant EphA1 can be employed to study its role in neural development, axon guidance, and synaptic plasticity.

6. Drug Discovery and Therapeutic Target Validation: Recombinant EphA1 is useful for screening potential inhibitors or activators of EphA1 signaling, aiding in the development of targeted therapies for diseases where EphA1 is dysregulated.

7. Standardization and Reproducibility: Using recombinant protein ensures a consistent and well-characterized reagent, which is essential for reproducible experimental results across different studies and laboratories.

In summary, Recombinant Human EphA1 is a versatile and essential reagent for investigating Eph receptor biology, with broad applications in cancer, vascular biology, immunology, neuroscience, and drug discovery.

Yes, recombinant human EphA1 can be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized and matches the assay’s requirements. Recombinant proteins are commonly used as standards in ELISA protocols for quantitative analysis of target proteins such as EphA1.

Key considerations for using recombinant EphA1 as an ELISA standard:

• Purity and Characterization: The recombinant EphA1 should be highly purified and its concentration accurately determined, ideally by methods such as HPLC or spectrophotometry.
• Standard Curve Preparation: Prepare a standard curve with serial dilutions of the recombinant EphA1 for each experiment. This curve is essential for quantifying unknown samples.
• Assay Compatibility: Confirm that the ELISA antibodies recognize both natural and recombinant forms of EphA1. Most commercial kits are validated to detect both forms, ensuring reliable quantification.
• Matrix Effects: Dilute the recombinant standard in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate calibration.
• Documentation: Follow the manufacturer’s instructions for reconstitution and dilution, as protocols may vary between lots and suppliers.

Limitations and best practices:

• Calibration: If the recombinant EphA1 is not mass-calibrated, it should be calibrated against a master standard to ensure consistency and accuracy.
• Validation: Run pilot experiments to validate the standard curve and confirm the linearity and sensitivity of your assay with the recombinant standard.
• Quality Assurance: Use a standard curve range appropriate for your expected sample concentrations, typically spanning the assay’s sensitivity limits.

Summary Table: Recombinant EphA1 as ELISA Standard

In conclusion, recombinant human EphA1 is suitable as a standard for ELISA quantification if it is properly characterized, compatible with the assay antibodies, and used according to best practices for standard curve preparation and calibration.

Recombinant Human EphA1 protein has been validated for several applications in published research, primarily in the context of cancer biology, signaling studies, and drug discovery. Key applications include:

• Cancer Research: EphA1 has been implicated in carcinogenesis and is associated with cancer metastasis and invasion in various cancers, including prostate, gastric, and liver cancer. Studies have shown that high expression of EphA1 correlates with aggressive tumor behavior and poor prognosis in these cancers.

• Signaling Pathway Analysis: EphA1 is involved in mediating developmental events, particularly in the nervous system, and plays a role in cell-cell communication and tissue homeostasis. It is used to study Eph/ephrin signaling pathways and their impact on cell migration, adhesion, and differentiation.

• Drug Discovery and Screening: Recombinant EphA1 is used in small molecule screening and kinase activity assays to identify potential inhibitors or modulators of EphA1 activity. This is particularly relevant for developing targeted therapies in cancers where EphA1 is overexpressed.

• Antibody Development and Validation: EphA1 recombinant protein is used for the generation and validation of antibodies, which are essential tools for immunohistochemistry (IHC), Western blotting (WB), flow cytometry (FC), and immunoprecipitation studies.

• Structure Determination: The protein is utilized in structural biology studies to determine the three-dimensional structure of EphA1, which aids in understanding its function and designing specific inhibitors.

• Bioassay Development: EphA1 is used to develop bioassays for studying receptor-ligand interactions, ligand binding, and downstream signaling events.

These applications highlight the versatility of recombinant Human EphA1 in both basic research and translational studies, particularly in the fields of oncology and developmental biology.

To reconstitute and prepare Recombinant Human EphA1 protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water to achieve your desired concentration, typically in the range of 0.1–1.0 mg/mL. The protein is usually supplied in a lyophilized form with a storage buffer of PBS, pH 7.4.

Step-by-step protocol:

• Reconstitution:

  • Allow the vial to equilibrate to room temperature before opening to minimize condensation.
  • Add sterile, distilled water directly to the vial to reach the recommended concentration (commonly 0.1–1.0 mg/mL).
  • Gently swirl or invert the vial to mix. Avoid vigorous shaking or vortexing, as this can cause foaming and protein denaturation.
  • Let the solution sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the protein solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • Store aliquots at 4°C for short-term use (up to 1 week) or at –20°C to –80°C for long-term storage.
  • For enhanced stability during storage, especially at –20°C, consider adding 5–50% glycerol as a cryoprotectant.

• Preparation for Cell Culture:

  • Before adding to cell culture, dilute the reconstituted protein in sterile PBS or cell culture medium to the working concentration required for your experiment.
  • If the protein is to be used in functional assays, ensure the final buffer is compatible with your cell culture system (e.g., isotonic, serum-free if required).
  • Filter-sterilize the final working solution using a 0.22 μm filter if sterility is critical.

Additional notes:

• The protein is typically supplied at high purity (>95%) and low endotoxin (<1 EU/μg), making it suitable for cell-based assays.
• Always consult the specific product datasheet for any unique instructions regarding reconstitution or buffer compatibility, as formulations may vary slightly between suppliers.
• Avoid repeated freeze-thaw cycles, as this can lead to protein aggregation or loss of activity.

Summary Table:

This protocol ensures the recombinant EphA1 protein is properly reconstituted and ready for reliable use in cell culture experiments.