Recombinant Human TRAIL R3

Recombinant human TRAIL-R3 serves several important research applications, particularly in understanding TRAIL signaling biology and developing cancer therapeutics.

Structural and Functional Characteristics

TRAIL-R3 is a unique member of the TRAIL receptor family that encodes a 299 amino acid protein with 58% and 54% overall identity to TRAIL-R1 and TRAIL-R2, respectively. The protein possesses high-affinity TRAIL-binding capability, with binding affinities comparable to other TRAIL receptors (Kd values ranging from 0.04–0.36 nM for high-affinity sites and 0.38–9.0 nM for low-affinity sites).

A critical distinguishing feature is that TRAIL-R3 lacks a cytoplasmic signaling domain and is glycosyl-phosphatidylinositol (GPI)-linked to the cell membrane. This structural difference makes it fundamentally different from death-inducing receptors like TRAIL-R1 and TRAIL-R2, as it cannot directly trigger apoptotic signaling despite binding TRAIL with high affinity.

Research Applications

Decoy Receptor Studies: TRAIL-R3 functions as a decoy receptor that can sequester TRAIL ligand without initiating apoptotic cascades. This property makes it valuable for investigating how decoy receptors modulate TRAIL-mediated cell death and for studying TRAIL resistance mechanisms in cancer cells.

Tissue-Specific Expression Analysis: TRAIL-R3 shows restricted expression patterns, with transcripts detectable primarily in peripheral blood lymphocytes and spleen. Using recombinant TRAIL-R3 allows researchers to examine tissue-specific TRAIL signaling and immune cell responses.

Cancer Therapy Development: TRAIL-R3 is highly expressed in many primary gastrointestinal cancers, making recombinant TRAIL-R3 useful for investigating how this receptor contributes to TRAIL resistance in these malignancies. Understanding TRAIL-R3's role is essential for developing strategies to overcome therapeutic resistance.

Competitive Binding and Inhibition Studies: Recombinant TRAIL-R3 can be used to create soluble fusion proteins that competitively inhibit TRAIL-mediated activities, providing a tool for dissecting TRAIL signaling pathways and validating therapeutic targets.

Recombinant Human TRAIL R3 can be used as a standard for quantification or calibration in ELISA assays, provided it is appropriately validated and matched to your assay system.

Key considerations and supporting details:

• ELISA kits for TRAIL R3/TNFRSF10C are routinely calibrated using recombinant human TRAIL R3 as the standard. For example, the R&D Systems DuoSet ELISA kit specifically states that it is calibrated against a highly purified recombinant human TRAIL R3/Fc chimera, and the standard curve is generated using this recombinant protein. This is a common practice in commercial and custom ELISA development.

• Both natural and recombinant TRAIL R3 are recognized by validated ELISA kits, and the quantification is based on a standard curve generated from the recombinant protein. This allows for accurate measurement of TRAIL R3 in biological samples, assuming the recombinant standard is structurally and immunologically similar to the native protein.

• Best practices for use as a standard:

  • Ensure the recombinant TRAIL R3 is of high purity and its concentration is accurately determined (e.g., by BCA or absorbance at 280 nm).
  • The recombinant standard should be reconstituted and diluted in a buffer that matches the sample matrix as closely as possible to minimize matrix effects.
  • Prepare a fresh standard curve for each assay run, using serial dilutions of the recombinant protein.
  • Validate that the recombinant standard produces a parallel response to endogenous TRAIL R3 in your sample matrix (parallelism test), confirming that quantification is accurate across the expected concentration range.

• Carrier protein considerations: Some protocols recommend using recombinant protein with a carrier (e.g., BSA) for stability, especially for use as an ELISA standard, while carrier-free forms may be preferred for other applications. Choose the format that best matches your assay requirements.

• Documentation: Always refer to the Certificate of Analysis (CoA) for the recombinant protein to confirm its concentration and any specific instructions for use as a standard.

In summary:
You can use recombinant human TRAIL R3 as a standard for ELISA quantification, provided it is validated for your assay and prepared according to best practices for standard curve generation and matrix matching. This approach is widely accepted and used in both commercial kits and custom assay development.

Recombinant Human TRAIL R3 (TNFRSF10C) has been validated for several key applications in published research, primarily as a tool to study TRAIL-mediated apoptosis, receptor-ligand interactions, and decoy receptor function.

Validated applications include:

• Immunocytochemistry (ICC): Used to detect TRAIL R3 expression in cells, allowing localization and quantification of the receptor.
• Cell Culture Studies: Applied to investigate the protective effect of TRAIL R3 against TRAIL-induced apoptosis in various cancer cell lines and to study receptor modulation by chemotherapeutic agents.
• Neutralization Assays: Recombinant TRAIL R3/Fc chimera is used to neutralize TRAIL activity, confirming its role as a decoy receptor that inhibits TRAIL-induced apoptosis.
• Western Blot: Utilized to detect TRAIL R3 protein in cell lysates and to analyze changes in expression following experimental treatments.
• Flow Cytometry: Employed to quantify cell surface expression of TRAIL R3 and to assess its involvement in apoptosis and immune cell regulation.
• Protein-Protein Interaction Analyses: Used to study binding affinity and specificity between TRAIL and its receptors, including TRAIL R3.
• Therapeutic Development Research: Investigated as a potential antagonist in cancer therapy and immune modulation, given its ability to inhibit TRAIL-induced cell death.

Supporting details:

• TRAIL R3 is a membrane-bound decoy receptor lacking the intracellular death domain, thus it does not transduce apoptotic signals but instead protects cells from TRAIL-induced apoptosis by competitive ligand binding.
• Recombinant TRAIL R3 is frequently used in cancer biology to dissect the mechanisms of TRAIL resistance and to evaluate the therapeutic potential of TRAIL pathway modulation.
• Flow cytometry and immunocytochemistry are standard methods for assessing TRAIL R3 expression and function in immune and cancer cell populations.
• Neutralization assays with recombinant TRAIL R3/Fc are critical for confirming its decoy activity and for studying the balance between death and decoy receptors in apoptosis regulation.

Additional relevant information:

• TRAIL R3 has been used in studies of inflammation, tissue regeneration, and immune homeostasis, particularly in models of colitis and cancer.
• Its restricted expression pattern (mainly in lymphocytes and spleen) makes it a valuable marker in immunological research.
• Recombinant TRAIL R3 is not intended for diagnostic or therapeutic use in humans but is widely employed in preclinical and mechanistic studies.

In summary, Recombinant Human TRAIL R3 is validated for ICC, cell culture, neutralization, Western blot, flow cytometry, and protein interaction studies, with broad utility in cancer, immunology, and cell biology research.

To reconstitute and prepare Recombinant Human TRAIL R3 protein for cell culture experiments, use sterile technique and follow these general steps, adapting as needed based on your specific product’s datasheet:

1. Equilibrate the lyophilized protein vial and your reconstitution buffer (commonly sterile PBS, pH 7.4) to room temperature before opening to minimize condensation.

2. Centrifuge the vial briefly to collect all lyophilized material at the bottom.

3. Reconstitute the protein:

   • Add sterile PBS to achieve the desired concentration (a common starting point is 100 μg/mL, but always check your product’s Certificate of Analysis for the recommended concentration).
   • Gently mix by inversion or slow pipetting. Avoid vigorous vortexing to prevent protein denaturation.
   • If the protein does not fully dissolve, allow it to sit at room temperature for 15–30 minutes with gentle agitation. If visible particulates remain, extend mixing up to 2 hours.

4. Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade protein activity.

5. Storage:

   • Store aliquots at 4–8 °C for short-term use (2–7 days).
   • For long-term storage, keep aliquots at –20 °C or below (up to 3 months or as specified by your supplier).

6. Preparation for cell culture:

   • Before adding to cell cultures, dilute the protein to the working concentration using sterile PBS or cell culture medium without carrier proteins or serum, unless otherwise specified.
   • Filter sterilize the final working solution if sterility is a concern and the protein is compatible with filtration.

Additional notes:

• Always refer to the specific product datasheet or Certificate of Analysis for any unique instructions, as formulations and recommended buffers may vary.
• If your protein is supplied with stabilizers (e.g., trehalose, mannitol, Tween-80), these are generally compatible with cell culture but confirm compatibility with your assay.
• If the protein is not validated for bioactivity, consider running a pilot experiment to confirm functional activity in your system.

Summary of best practices:

• Use sterile PBS for reconstitution unless otherwise specified.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Store reconstituted protein at 4–8 °C (short-term) or –20 °C (long-term).
• Dilute to working concentration in sterile buffer or medium before use in cell culture.

If your application is highly sensitive to endotoxin, confirm the endotoxin level is suitable for cell culture (<1 EU/μg is typical for recombinant proteins used in cell-based assays).