Recombinant Human TRAIL R2

Recombinant Human TRAIL R2 is widely used in research applications to study apoptosis, cancer biology, and immune signaling because it is a key receptor mediating TRAIL-induced cell death and can be manipulated to probe or modulate these pathways.

Key scientific reasons to use Recombinant Human TRAIL R2:

• Functional Apoptosis Receptor: TRAIL R2 (also known as DR5 or TNFRSF10B) is a principal receptor for the death ligand TRAIL, triggering apoptosis in many cancer cell types upon ligand binding. Recombinant TRAIL R2 enables controlled studies of ligand-receptor interactions, downstream signaling, and cell fate decisions.

• Cancer Research Applications: TRAIL R2 is highly expressed in various cancers and is a target for therapeutic strategies aiming to selectively induce apoptosis in tumor cells while sparing normal cells. Recombinant forms allow for in vitro and in vivo modeling of these processes, screening of agonists/antagonists, and mechanistic studies.

• Neutralization and Blocking Studies: The extracellular domain of recombinant TRAIL R2 can bind TRAIL and inhibit its function, making it useful for blocking experiments to dissect TRAIL-dependent pathways or to validate specificity in apoptosis assays.

• Synergy and Combination Therapy Research: Recombinant TRAIL R2 is used to investigate synergistic effects with TRAIL or TRAIL-R2-specific antibodies, which can enhance cancer cell killing and inform combination therapy strategies.

• Immune and Inflammatory Signaling: TRAIL-R2 signaling is implicated in immune regulation and inflammation, including NF-κB activation and cytokine production. Recombinant TRAIL R2 is valuable for studying these non-apoptotic functions and their impact on tumor microenvironment and immune cell behavior.

• Receptor Expression and Resistance Mechanisms: Recombinant TRAIL R2 is essential for exploring mechanisms of TRAIL resistance, receptor expression patterns, and the development of sensitization protocols for resistant cancer cells.

Typical research applications include:

• Apoptosis assays (e.g., ligand binding, receptor activation, cell death quantification)
• Screening of TRAIL agonists, antagonists, or antibodies
• Mechanistic studies of death receptor signaling
• Investigating tumor cell sensitivity and resistance to TRAIL-based therapies
• Elucidating immune cell interactions and cytokine responses

Best practices: Use recombinant TRAIL R2 in well-controlled experimental systems, validate specificity with appropriate controls (e.g., TRAIL-R1, blocking antibodies), and consider both apoptotic and non-apoptotic signaling outcomes for comprehensive analysis.

In summary, Recombinant Human TRAIL R2 is a versatile tool for dissecting TRAIL-mediated signaling, apoptosis, and cancer biology, and for developing and validating targeted therapies.

Recombinant Human TRAIL R2 can be used as a standard for quantification or calibration in ELISA assays, provided it is immunologically recognized by the assay antibodies and its concentration is value-assigned within the ELISA system.

Most commercial ELISA kits for TRAIL R2 (also known as CD262, DR5, TNFRSF10B) are designed to detect both natural and recombinant forms of the protein, and their standard curves are often generated using recombinant TRAIL R2 as the calibrator. The key requirements are:

• The recombinant TRAIL R2 must be recognized by the capture and detection antibodies used in your ELISA system.
• The protein should be properly diluted to fall within the validated standard curve range of your assay.
• The concentration assigned to the recombinant standard should be determined by measurement in the ELISA itself, not solely by the mass on the vial label, due to possible differences in immunoreactivity and dilution errors.

Best Practices:

• Prepare a standard curve using serial dilutions of recombinant TRAIL R2 in the same buffer or matrix as your samples.
• Validate that the recombinant standard produces a linear and reproducible response in your ELISA system.
• If using a recombinant protein not supplied with your kit, assign its value by running it in parallel with a validated standard, as mass values may differ due to lot-to-lot variability and immunoreactivity.
• Ensure the recombinant protein covers the expected concentration range of your samples and matches the sensitivity of your assay.

Limitations:

• Some ELISA kits are validated only for native TRAIL R2 and may not recognize all recombinant forms, especially if the recombinant protein lacks certain post-translational modifications or domains.
• Always consult the technical documentation for your specific ELISA kit to confirm compatibility with recombinant standards.

Summary Table: Use of Recombinant TRAIL R2 as ELISA Standard

In conclusion: Recombinant Human TRAIL R2 is suitable as an ELISA standard if it is immunologically compatible and its concentration is properly assigned within your assay system.

Recombinant Human TRAIL R2 (also designated TNFRSF10B or DR5) has been validated for several key applications in published research:

Primary Research Applications

Bioassay and Functional Studies

TRAIL R2 recombinant proteins have been extensively used in bioassay applications to assess apoptotic signaling pathways. These functional assays measure the receptor's ability to mediate TRAIL-induced apoptosis and evaluate the effects of various compounds on death receptor signaling. The protein has been particularly valuable in studying TRAIL-mediated cytotoxicity in cancer cell lines, including investigations of apoptosis regulation in Burkitt's lymphoma and other malignancies.

Neutralization and Blocking Assays

A critical application involves using TRAIL R2 as a neutralizing agent to inhibit TRAIL-mediated cytotoxicity. Recombinant TRAIL R2-Fc fusion proteins function as competitive inhibitors, blocking the interaction between soluble TRAIL and cell surface receptors. This application has been instrumental in elucidating the specific contributions of TRAIL R2 versus TRAIL R1 in apoptotic pathways and in studying immune cell regulation.

Immunological and Diagnostic Applications

The protein has been validated for use in ELISA assays, Western blotting, and flow cytometry applications. These techniques enable detection and quantification of TRAIL receptor expression on cell surfaces and assessment of receptor-ligand interactions in various cell types and tissues.

Therapeutic Development Context

Beyond basic research, recombinant TRAIL R2 has supported the development of TRAIL-R2-specific agonistic antibodies and combination therapeutic strategies for cancer treatment. Studies have demonstrated that combining recombinant TRAIL with TRAIL-R2-specific antibodies can synergistically enhance death-inducing signaling complex formation, leading to improved cancer cell killing compared to either agent alone.

To reconstitute and prepare Recombinant Human TRAIL R2 protein for cell culture experiments, follow these best-practice steps based on general recombinant protein handling protocols and TRAIL R2-specific recommendations:

1. Equilibrate Materials

• Allow the lyophilized protein vial and your chosen reconstitution buffer (commonly sterile distilled water or PBS) to reach room temperature before opening.

2. Collect Lyophilized Protein

• Briefly centrifuge or tap the vial to ensure all powder is at the bottom.

3. Add Reconstitution Buffer

• Add the recommended volume of sterile buffer to achieve your desired concentration. For TRAIL R2, protocols often suggest reconstituting in sterile water or PBS, but always check the product datasheet or Certificate of Analysis for specific instructions.
• For example, some protocols recommend not reconstituting below 100 μg/mL in ddH₂O for stability.

4. Dissolve Gently

• Let the vial stand at room temperature for 15–30 minutes to allow the protein to dissolve.
• Gently pipette up and down to mix. Do not vortex or shake vigorously, as this can denature the protein and reduce biological activity.

5. Inspect Solution

• The solution should be clear. If particulates remain, gently mix for up to 2 hours at room temperature.

6. Aliquot and Storage

• Use the reconstituted protein immediately for experiments, or aliquot into small volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at –20 °C or –80 °C for long-term storage. Avoid multiple freeze-thaw cycles to preserve activity.

7. Preparation for Cell Culture

• For cell culture, dilute the reconstituted stock to the working concentration using sterile cell culture medium. It is advisable to include a carrier protein (such as 0.1% BSA or 5% FBS) in the dilution buffer to minimize adsorption and stabilize the protein, especially at low concentrations.
• Filter-sterilize the final working solution if sterility is required and the buffer composition allows.

Key Technical Notes:

• Do not vortex or use harsh agitation at any step.
• Carrier protein is not needed for initial reconstitution but is recommended for long-term storage or when diluting to low concentrations.
• Always consult the specific product datasheet for any unique requirements, as protein tags, expression systems, or formulation may affect solubility and stability.

Example Protocol (Generic for TRAIL R2):

1. Add 100 μL sterile water or PBS to the lyophilized vial (for a typical 100 μg vial, adjust as needed for your concentration).2. Let stand at room temperature for 20 minutes.3. Gently pipette to mix until fully dissolved.4. Aliquot and store unused portions at –20 °C.5. For cell culture, dilute to working concentration in medium containing 0.1% BSA or 5% FBS.

References:

If you have a specific TRAIL R2 construct (e.g., Fc chimera, tag, or carrier-free), always verify the recommended buffer and concentration in the product documentation, as solubility and stability can vary.