Recombinant Human TRAIL R4

Recombinant Human TRAIL R4 is primarily used in research to investigate the regulatory mechanisms of apoptosis and cell survival, particularly in the context of cancer biology. TRAIL R4 (also known as Decoy Receptor 2, DcR2) is a member of the TRAIL receptor family that modulates the effects of TRAIL-induced cell death and survival signaling pathways.

Key scientific applications and rationale for using recombinant TRAIL R4 include:

• Deciphering Apoptosis Regulation: TRAIL R4 acts as a decoy receptor, inhibiting TRAIL-induced apoptosis by competing for TRAIL binding and preventing activation of death receptors TRAIL-R1 and TRAIL-R2. This makes recombinant TRAIL R4 valuable for dissecting the balance between pro-apoptotic and anti-apoptotic signaling in various cell types, especially cancer cells.

• Studying Tumor Resistance Mechanisms: Overexpression or upregulation of TRAIL R4 has been linked to increased resistance to TRAIL-mediated apoptosis in tumor cells, contributing to tumor growth and survival. Recombinant TRAIL R4 can be used to model these resistance mechanisms and to screen for agents that overcome TRAIL resistance.

• Exploring Non-Apoptotic Signaling: TRAIL R4 is involved in the regulation of non-apoptotic, pro-survival pathways such as NF-κB, AKT, and ERK signaling. Using recombinant TRAIL R4 allows researchers to investigate how these pathways are modulated in response to TRAIL and how they contribute to cell fate decisions.

• Immune Surveillance and Microenvironment Studies: TRAIL R4 plays a role in immune surveillance and the protection of non-malignant cells from TRAIL-induced apoptosis. Recombinant TRAIL R4 can be used to study its physiological function in normal tissues and its impact on the tumor microenvironment.

• Functional Assays and Pathway Analysis: Recombinant TRAIL R4 is essential for in vitro assays such as:

  • Apoptosis assays (e.g., caspase activation, cell viability)
  • Clonogenic survival assays
  • Western blotting for pathway activation (e.g., NF-κB, ERK, AKT)
  • Protein-protein interaction studies

• Therapeutic Target Validation: By manipulating TRAIL R4 levels or activity in cell models, researchers can validate its potential as a therapeutic target for sensitizing tumors to TRAIL-based therapies or for protecting normal tissues during treatment.

Best Practices:

• Use recombinant TRAIL R4 in combination with recombinant TRAIL and other TRAIL receptors to map signaling outcomes.
• Employ dose-response and time-course experiments to characterize TRAIL R4’s effects on apoptosis and survival pathways.
• Validate findings with genetic knockdown or overexpression approaches for mechanistic insights.

In summary, recombinant human TRAIL R4 is a critical tool for elucidating the complex interplay between cell death and survival in cancer and normal cells, advancing both basic research and therapeutic development.

Suitability for ELISA Calibration

Recombinant human TRAIL R4 can be used as a standard for ELISA quantification, but with important considerations regarding accuracy and validation.

Technical Considerations

Immunological Recognition

Recombinant TRAIL R4 proteins are recognized by sandwich ELISA systems designed to detect both natural and recombinant forms of the target. The assay antibody pairs will bind to recombinant protein, allowing it to generate a measurable signal on the standard curve.

Potential Accuracy Issues

However, there are several factors that can affect the reliability of recombinant proteins as calibration standards:

• Large dilution steps: Converting from typical recombinant protein concentrations (μg/mL) to ELISA standard ranges (pg/mL) requires substantial dilutions, which can introduce cumulative measurement errors
• Lot-to-lot variability: The immunologically recognizable mass of recombinant protein may vary between manufacturing lots, causing apparent concentration differences when measured by ELISA compared to the vial label
• Mass discrepancies: Values calculated by ELISA may differ from the mass reported on the protein vial due to these immunological variations

Best Practices for Standard Preparation

For optimal calibration accuracy, purified proteins are preferred for standard curve preparation. If using recombinant proteins as standards, consider these approaches:

• Value assignment: Rather than relying on the vial label concentration, measure your recombinant protein in the ELISA system and use that measured value to establish control levels
• Semi-purification: If necessary, semi-purify recombinant protein in your laboratory and measure concentration using complementary techniques such as HPLC
• Validation: Ensure your recombinant standard performs consistently across multiple assay runs and operators

Application Limitations

Note that recombinant proteins designated as ELISA standards are specifically validated for calibration purposes and are not recommended for bioassay applications, which require different validation criteria.

Recombinant Human TRAIL R4 (also known as TNFRSF10D, DcR2, or CD264) has been validated in published research for several key applications, primarily in the context of cancer biology and apoptosis signaling. The most common validated applications include:

• Cell-based functional assays: Recombinant TRAIL R4 is used to study its regulatory role in apoptosis and non-apoptotic signaling pathways in various human cancer cell lines. For example, knockdown and overexpression studies have elucidated how TRAIL R4 modulates sensitivity to TRAIL-induced apoptosis, clonogenic survival, and the expression of anti-apoptotic proteins in cell lines such as Colo357 and MDA-MB-231.

• Western blotting: TRAIL R4 protein and its downstream signaling effects are frequently analyzed by Western blot to assess changes in protein expression and pathway activation following manipulation of TRAIL R4 levels or treatment with recombinant TRAIL.

• ELISA and immunoassays: Recombinant TRAIL R4 is used as a standard or analyte in ELISA kits to quantify TRAIL R4 levels in human serum, plasma, or cell culture supernatants. It is also used to validate antibody specificity in sandwich immunoassays and related formats.

• Flow cytometry: Antibodies against TRAIL R4, sometimes validated using recombinant protein, are used to detect cell surface expression of TRAIL R4 on various cell types by flow cytometry.

• In vitro binding and competition assays: Recombinant TRAIL R4 is used to study ligand-receptor interactions, including its decoy function in sequestering TRAIL and modulating apoptosis signaling.

• Cancer biology and therapeutic research: Recombinant TRAIL R4 is employed in studies investigating its role as a decoy receptor that inhibits TRAIL-induced apoptosis in tumor cells, as well as its potential as a therapeutic target or biomarker in cancer.

Additional research contexts:

• Studies have used recombinant TRAIL R4 to explore its physiological role in protecting specific cell types (e.g., hepatic stellate cells) from apoptosis.
• Recombinant TRAIL R4 has been used in preclinical models to investigate its impact on immune cell survival and inflammatory signaling.

Summary Table: Applications of Recombinant Human TRAIL R4 in Published Research

In summary, recombinant human TRAIL R4 is a versatile tool in apoptosis, cancer, and immunology research, validated for use in functional cell assays, protein detection (Western blot, ELISA), flow cytometry, and mechanistic studies of TRAIL signaling.

To reconstitute and prepare Recombinant Human TRAIL R4 protein for cell culture experiments, first confirm the specific formulation and lyophilization buffer from the product datasheet. The following protocol summarizes best practices based on current technical literature and standard recombinant protein handling:

1. Centrifuge the vial:
Briefly centrifuge the lyophilized protein vial to ensure all material is at the bottom before opening.

2. Reconstitution:

• Add sterile distilled water or sterile PBS (pH 7.4) to the vial.
• Typical reconstitution concentrations are 0.1–0.5 mg/mL for TRAIL R4.
• Gently mix by swirling or inverting; avoid vigorous pipetting or vortexing to prevent protein denaturation.

3. Stabilization (optional but recommended):

• For improved stability, especially for storage or dilute working solutions, add a carrier protein such as 0.1–1% BSA, 5% HSA, 10% FBS, or 5% trehalose.
• This is particularly important if the protein will be stored after reconstitution or used at low concentrations.

4. Incubation:

• Allow the protein to fully dissolve at room temperature for 15–30 minutes with gentle agitation.

5. Aliquoting and Storage:

• Aliquot the reconstituted protein to avoid repeated freeze/thaw cycles.
• Store at –20°C for up to 3 months or at 2–8°C for up to 1 week.
• For long-term storage, consider adding glycerol (final 50%) if compatible with your downstream application.

6. Preparation for Cell Culture:

• Before use, dilute the protein to the desired working concentration in cell culture medium.
• If carrier protein was used during reconstitution, ensure it is compatible with your assay and cell type.

Key technical notes:

• Avoid vigorous mixing to prevent aggregation or loss of activity.
• Minimize freeze/thaw cycles by aliquoting.
• Always use sterile technique to prevent contamination.

Example protocol for 100 μg lyophilized TRAIL R4:

1. Centrifuge vial briefly.2. Add 200 μL sterile distilled water (for 0.5 mg/mL final concentration).3. Gently swirl to dissolve.4. Add 0.1% BSA if desired for stabilization.5. Incubate at room temperature for 20 minutes.6. Aliquot and store at –20°C.

References:

• Biotinylated Recombinant Human TRAIL-R4/CD264 Protein datasheet
• Recombinant Proteins Support—Getting Started
• How to Reconstitute Lyophilized Proteins

If your product datasheet specifies a different buffer or concentration, always follow the manufacturer’s instructions for optimal results.