Recombinant Rat EphA5

Recombinant Rat EphA5 is valuable in research applications because it enables precise investigation of EphA5’s biological functions, signaling mechanisms, and therapeutic potential, particularly in cancer biology and neurobiology.

Key scientific reasons to use recombinant rat EphA5 include:

• Functional Studies: Recombinant EphA5 allows for controlled in vitro and in vivo experiments to dissect its role in cell signaling, cell cycle regulation, and DNA damage repair. EphA5 has been identified as a regulator of cell cycle checkpoints and DNA repair, especially in response to ionizing radiation, making it a target for studies on radioresistance and cancer therapy.

• Therapeutic Target Validation: EphA5 is expressed in various solid tumors and is implicated in tumor proliferation, apoptosis inhibition, and resistance to radiotherapy. Recombinant protein is essential for validating antibody specificity, screening for inhibitors, and developing targeted therapies such as antibody-drug conjugates (ADCs).

• Protein-Protein Interaction Assays: Recombinant rat EphA5 is used in binding assays, such as ELISA, to study interactions with ephrin ligands (e.g., Ephrin-A2), which are critical for understanding Eph/ephrin signaling pathways in development, cancer, and tissue regeneration.

• Cross-Species Conservation: The extracellular domain of EphA5 is highly conserved across species, including rat and human, supporting its use in translational studies and preclinical models.

• Biomarker Development: EphA5’s role as a biomarker for radioresistance and tumor progression can be explored using recombinant protein in diagnostic assay development.

• Cellular Communication Research: EphA5 is involved in mediating cell-cell interactions, especially in the nervous system and bone marrow microenvironment, making recombinant protein useful for studying developmental biology and tissue engineering.

Best practices for using recombinant rat EphA5:

• Employ in functional ELISA assays to confirm ligand binding activity.
• Use in Western blot and immunoassays to validate antibody specificity and protein expression.
• Apply in cell-based assays to investigate downstream signaling and cellular responses.

In summary, recombinant rat EphA5 is a critical tool for mechanistic studies, therapeutic development, and biomarker research in oncology, neurobiology, and cell signaling.

Yes, recombinant rat EphA5 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system. Recombinant proteins are commonly used as standards in ELISA to generate calibration curves for quantifying target analytes in biological samples.

Key considerations for use:

• Assay Compatibility: The ELISA must be designed to recognize both natural and recombinant rat EphA5. Many commercial rat EphA5 ELISA kits specify that their antibodies detect both forms, allowing recombinant EphA5 to serve as a standard.
• Standard Preparation: The recombinant protein should be reconstituted and diluted according to the kit or assay protocol to generate a standard curve covering the expected concentration range in your samples.
• Protein Formulation: Recombinant EphA5 is often supplied with or without carrier proteins (e.g., BSA). For ELISA calibration, formulations with BSA are generally recommended for stability, unless BSA interferes with your assay.
• Validation: Confirm that the recombinant EphA5 standard yields a linear, reproducible standard curve and that its signal is comparable to endogenous EphA5 in your sample matrix. This ensures accurate quantification.

Best Practices:

• Use the recombinant standard provided or recommended by your ELISA kit manufacturer, as it will have been validated for that specific assay.
• If using a recombinant standard from another source, perform a parallelism test to verify that the standard curve accurately reflects the behavior of endogenous EphA5 in your samples.
• Store and handle the recombinant protein according to manufacturer instructions to maintain stability and activity.

Summary Table: Recombinant Rat EphA5 as ELISA Standard

In conclusion, recombinant rat EphA5 is suitable as a standard for ELISA quantification, provided it is validated for your specific assay system and protocol.

Recombinant Rat EphA5 has been validated in published research for several key applications, primarily in studies of cancer biology, neurobiology, and molecular targeting. The most prominent validated applications include:

• Antibody-based detection assays such as ELISA and Western blot
• Preclinical cancer therapy models (including antibody-drug conjugate efficacy and toxicology)
• Functional studies of cell signaling, DNA damage response, and radioresistance
• In vivo imaging and xenograft models
• Protein targeting in neurobiological research

Supporting Details

1. Detection Assays

• Recombinant rat EphA5 has been used as a standard or antigen in direct ELISAs and Western blots to detect EphA5 protein and validate antibody specificity.

2. Preclinical Cancer Therapy

• Recombinant EphA5 is central to the development and validation of antibody-drug conjugates (ADCs) targeting EphA5, such as MBRC-101. These studies include:
  • Efficacy testing in rat and mouse xenograft models of human cancers (e.g., lung cancer, triple-negative breast cancer).
  • Toxicology studies in rats to assess safety and pharmacokinetics of EphA5-targeted ADCs.
  • Immunohistochemistry (IHC) and flow cytometry to confirm EphA5 expression in tumor models.

3. Functional Cell Biology

• Recombinant EphA5 has been used to study its role in cell cycle regulation and DNA damage repair following ionizing radiation, particularly in lung cancer models. These studies demonstrate EphA5’s involvement in radioresistance and its potential as a therapeutic target.

4. In Vivo Imaging

• Radiolabeled recombinant EphA5 antibodies have been used for in vivo imaging in rat xenograft models, enabling visualization and quantification of EphA5 expression in tumors.

5. Neurobiological Research

• Recombinant EphA5 has been applied in studies of brain development and function, including protein targeting strategies to manipulate EphA5 activity in vivo for learning and memory research.

Additional Validated Uses

• Ligand binding studies: Recombinant EphA5 has been used to characterize ligand specificity and binding dynamics, including peptide interaction assays.
• Protein targeting: In vivo application of recombinant fusion proteins to modulate EphA5 function in the rat brain.

Summary Table

In summary, recombinant rat EphA5 is a validated tool for molecular detection, cancer therapy development, functional cell biology, imaging, and neurobiological research in published literature.

To reconstitute and prepare Recombinant Rat EphA5 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) to achieve a stock concentration of at least 100 µg/mL.

Detailed protocol and best practices:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS directly to the vial to reach the desired concentration (≥100 µg/mL).
• Gently mix by pipetting up and down or by slow inversion. Avoid vigorous shaking or vortexing, as this can denature the protein.
• Allow the protein to fully dissolve at room temperature for 15–30 minutes with gentle agitation.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
• For short-term storage (up to 1 week), keep the reconstituted protein at 2–8 °C.
• For long-term storage, dilute the protein with a carrier protein solution (e.g., 0.1% BSA in PBS), aliquot, and store at –20 °C or –80 °C. If using in serum-free or animal experiments, use a non-protein stabilizer such as trehalose instead of BSA.
• Before use in cell culture, dilute the stock solution to the working concentration using cell culture medium. If the experiment is sensitive to animal proteins, ensure the diluent is compatible with your assay system.

Additional notes:

• The recombinant rat EphA5 protein is typically expressed as an Fc chimera and is lyophilized from a sterile-filtered PBS solution.
• Always consult the specific Certificate of Analysis (CoA) for your protein batch for any unique instructions.
• Avoid repeated freeze-thaw cycles to maintain protein integrity and activity.

This protocol ensures the protein remains stable and functional for cell culture applications.