Recombinant Human Death receptor-6 (DR6)

Recombinant Human Death receptor-6 (DR6) is a valuable tool in research applications due to its critical roles in apoptosis, neurodegeneration, immune regulation, and disease modeling.

DR6 is a member of the TNF receptor superfamily, containing a death domain that mediates apoptotic signaling and interacts with key adaptor proteins such as TRADD. Its functions include:

• Neuronal apoptosis and axonal degeneration: DR6 interacts with amyloid precursor protein (APP) and p75^NTR^, promoting neuronal cell death and axonal pruning, which is relevant for studying neurodegenerative diseases like Alzheimer’s disease. Elevated DR6 expression is observed in affected brain regions, and manipulating DR6 can alter neurotoxicity and cell survival.

• Immune regulation: DR6 is highly expressed in lymphoid tissues and regulates CD4^+^ T cell proliferation, T helper cell differentiation, and B cell expansion. Disruption or antagonism of DR6 affects T cell responses, antibody production, and can modulate autoimmune disease progression, such as in lupus and multiple sclerosis models.

• Cancer and apoptosis research: DR6 is implicated in mitochondria-dependent apoptosis and can serve as a biomarker for certain tumors, including gliomas and ovarian cancer. Its signaling pathways (NF-κB, JNK) are relevant for studying cell survival and death mechanisms in cancer biology.

• Dendritic cell maturation and anti-tumor immunity: DR6 influences dendritic cell survival and maturation, impacting immune responses and offering potential for anti-tumor therapeutic strategies.

• Disease modeling and therapeutic target validation: Recombinant DR6 enables mechanistic studies, screening of antagonists or agonists, and validation of therapeutic strategies targeting DR6 in neurodegeneration, autoimmunity, and cancer.

Typical research applications include:

• Functional assays of apoptosis and cell signaling.
• Disease modeling in neurodegeneration and autoimmunity.
• Screening for therapeutic antibodies or small molecules targeting DR6.
• Biomarker studies in cancer and neurological disorders.

Using recombinant DR6 provides a controlled, reproducible system for dissecting its molecular mechanisms and evaluating potential interventions in relevant disease contexts.

Yes, recombinant human Death Receptor 6 (DR6) can be used as a standard for quantification and calibration in ELISA assays. DR6, also known as TNFRSF21 or CD358, is a type I transmembrane protein and member of the TNF receptor superfamily, and recombinant versions of this protein are specifically designed and validated for use in immunoassay applications.

Standard Preparation and Calibration

Recombinant human DR6 proteins are commercially available as calibration standards in high purity formats. These standards are typically supplied in multiple vials, with quantities sufficient to run standards in duplicate across multiple assay plates. The recombinant protein can be diluted serially to establish calibration curves for sandwich ELISA formats.

Validated Concentration Ranges

When using recombinant DR6 as a standard, typical quantitative ranges for sandwich ELISA assays span from approximately 0.4 to 6.4 ng/mL or 15.625 to 1000 pg/mL, depending on the specific assay configuration. The selection of your concentration range should be validated based on your sample types and expected protein levels.

Important Considerations for Accurate Quantification

Calibration against master calibrators: If you are using recombinant DR6 as a quality control or secondary standard, be aware that commercial ELISA kits are typically calibrated against master calibrators established during kit development. When using retail recombinant proteins as controls, expect approximately ±25% recovery variation between the stated mass on the vial and the measured concentration in your ELISA. This discrepancy arises from large dilution steps (typically from μg/mL to pg/mL ranges) and differences in immunologically recognizable mass between protein lots.

Validation requirements: For optimal accuracy, it is recommended to value-assign the recombinant DR6 standard based on ELISA measurement rather than relying solely on the manufacturer's stated concentration. This approach ensures consistency with your specific assay conditions and antibody pairs.

Antibody pair compatibility: Ensure that your capture and detection antibodies are validated as a matched pair for DR6/TNFRSF21 detection, as this is critical for sandwich ELISA performance and accurate standard curve generation.

Recombinant Human Death Receptor-6 (DR6) has been validated in published research for applications in cell signaling studies, neurobiology, cancer biology, immunology, and as a potential therapeutic and diagnostic target.

Key validated applications include:

• Cell signaling and apoptosis assays: DR6 has been used to study apoptosis and cell survival pathways in various cell types, including neurons, HeLa cells, and HEK293T cells. It mediates both mitochondria-dependent apoptosis and NF-κB pathway activation, depending on cellular context.
• Neurodegeneration and axonal pruning: Recombinant DR6 has been employed to investigate its interaction with amyloid precursor protein (APP), which is implicated in axonal degeneration and synapse loss, particularly relevant to Alzheimer's disease models.
• Cancer research: DR6 is involved in tumor cell extravasation and metastasis. Recombinant DR6 and its inhibitors (such as PEG-tAHP-DRI) have been validated in vitro and in vivo to block tumor cell-induced endothelial necroptosis and reduce metastasis in mouse models.
• Autoimmunity and immunology: DR6 has been studied as a regulator of T follicular helper (Tfh) cell expansion and activation, particularly in lupus-prone mice. Recombinant DR6 and DR6-specific antibodies have been validated for modulating immune responses and delaying disease progression in systemic lupus erythematosus (SLE) models.
• Oligodendrocyte survival and myelination: Recombinant DR6 has been used to study its role in oligodendrocyte apoptosis and myelination, with implications for demyelinating diseases such as multiple sclerosis.
• Protein-protein interaction studies: Structural and biochemical assays have validated recombinant DR6 for mapping its interactions with APP, p75^NTR^, TRADD, PSAP, and syndecan-1, using techniques such as crystallography and co-immunoprecipitation.
• Therapeutic antibody validation: Recombinant DR6 has been used as an antigen for generating and validating antagonistic and agonistic antibodies, which are then tested for their effects on DR6-mediated signaling and disease models.

Summary Table of Validated Applications

Additional notes:

• Recombinant DR6 is also being explored as a biomarker for certain cancers and neurological diseases, based on its elevated expression in patient samples.
• Functional studies often use recombinant DR6 in both in vitro (cell culture) and in vivo (mouse model) systems to validate its biological roles and therapeutic potential.

These applications are supported by a range of published studies employing recombinant DR6 in diverse experimental systems, confirming its utility in both basic and translational research.

To reconstitute and prepare Recombinant Human Death receptor-6 (DR6) protein for cell culture experiments, follow these best-practice steps based on protein handling guidelines and available product information:

• Storage (before reconstitution):
  Keep the lyophilized DR6 protein desiccated at –20°C to –70°C for long-term stability.

• Reconstitution:

  • Buffer: Use sterile, endotoxin-free water or sterile phosphate-buffered saline (PBS), unless the product datasheet specifies a different buffer.
  • Concentration: Common reconstitution concentrations are 0.1–1 mg/mL. For DR6, a typical starting point is 0.2 mg/mL in sterile PBS. Adjust as needed for your application.
  • Procedure:
    1. Briefly centrifuge the vial to collect all lyophilized powder at the bottom.
    2. Add the appropriate volume of buffer directly to the vial.
    3. Gently swirl or invert the vial to dissolve. Do not vortex or pipette up and down vigorously, as this may denature the protein.
    4. Allow the protein to fully dissolve at room temperature for 15–30 minutes. If necessary, gently mix by tilting or slow rotation.

• Aliquoting and Storage (after reconstitution):

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade protein activity.
  • Store aliquots at 2–8°C for up to one month, or at –20°C to –70°C for longer-term storage.
  • Avoid storing at –20°C if the buffer contains PBS, as this can cause precipitation; –70°C is preferable for long-term storage.

• Preparation for Cell Culture:

  • Dilute the reconstituted stock to the desired working concentration using sterile cell culture medium or buffer immediately before use.
  • Filter sterilize (0.22 μm) if sterility is required and the protein solution is compatible.
  • Confirm that the final buffer composition is compatible with your cell culture system (e.g., isotonic, pH 7.2–7.4, low endotoxin).

Additional Notes:

• Endotoxin: Ensure the endotoxin level is suitable for cell culture (<0.01 EU/μg is typical for sensitive applications).
• Handling: Always use aseptic technique to prevent contamination.
• Documentation: Refer to the specific product datasheet for any unique instructions regarding buffer composition, concentration, or handling.

Summary Table: DR6 Reconstitution and Preparation

Always consult the specific product datasheet for any unique requirements, as recombinant protein preparations can vary between suppliers.