Recombinant Human DR6

Recombinant Human DR6 (Death Receptor-6) offers several compelling advantages for research applications, particularly in studies involving immune regulation, neuronal biology, and disease modeling.

Research Applications and Functional Significance

DR6 is a death receptor expressed across most human tissues, with particularly high expression levels in the heart, brain, placenta, pancreas, thymus, and lymph nodes. This widespread distribution makes it relevant for investigating multiple biological systems and disease pathways.

Immune System Studies: DR6 plays a critical role in T cell biology. Targeted disruption of DR6 results in enhanced CD4+ T cell proliferation and T helper 2 differentiation in vitro, making recombinant DR6 valuable for studying T cell activation mechanisms, immune tolerance, and helper T cell differentiation pathways. This makes it particularly useful for research into autoimmune diseases and immune regulation.

Neuronal Research: DR6 is critical for neuronal apoptosis, particularly in response to amyloid-derived peptides, and plays an important role in normal cell body death and axonal pruning. This functionality makes recombinant DR6 an essential tool for neurodegenerative disease research, including studies on Alzheimer's disease pathology and motor neuron diseases.

Advantages as a Research Reagent

As a recombinant protein, DR6 provides the standard benefits inherent to recombinant systems: high purity, specificity, consistency, and customization capabilities. These characteristics make it suitable for use in established laboratory techniques such as ELISA, Western blotting, and immunohistochemistry, where it can serve as a positive control or standard. Additionally, recombinant DR6 can be utilized in protein-protein interaction studies to examine how DR6 engages with its ligands and downstream signaling partners, facilitating investigation of receptor-mediated cellular responses and disease mechanisms.

You can use recombinant Human DR6 as a standard for quantification or calibration in your ELISA assays, provided the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with the antibodies and detection system used in your assay.

Key considerations and best practices:

• Purity and Quantification: The recombinant DR6 should be highly purified, and its concentration must be accurately measured, ideally by methods such as HPLC or UV absorbance at 280 nm using a known extinction coefficient.
• Compatibility: The standard must be recognized by the capture and detection antibodies in your ELISA. Most commercial DR6 ELISA kits are validated to detect both natural and recombinant DR6, and protocols typically specify dilution series for recombinant DR6 standards.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant DR6 in the same buffer or diluent used for your samples to generate a standard curve. This allows for accurate quantification of DR6 in unknown samples.
• Validation: It is recommended to validate the recombinant standard in your specific assay system. Compare the standard curve generated with recombinant DR6 to that generated with natural DR6 (if available) to ensure parallelism and accurate quantification.
• Documentation: Record the lot number, purity, and quantification method for your recombinant DR6 standard to ensure reproducibility and traceability.

Limitations:

• Research Use Only: Recombinant standards are typically for research use and not for diagnostic procedures.
• Calibration: If absolute quantification is required, ensure your recombinant DR6 is calibrated against a mass-calibrated reference standard, as recommended for quantitative immunoassays.

In summary, recombinant Human DR6 is suitable as a standard for ELISA quantification if it is properly characterized and validated within your assay system.

Recombinant Human DR6 (Death Receptor 6, TNFRSF21) has been validated for several key applications in published research, primarily in the fields of neurobiology, immunology, and cancer biology.

Validated Applications:

• Protein-Protein Interaction Studies: Recombinant DR6 has been used to characterize its binding with amyloid precursor protein (APP), including structural studies and assays to elucidate the molecular basis of DR6/APP interaction. This interaction is implicated in axonal pruning and synapse inhibition in the nervous system.

• Cell Biology and Functional Assays: DR6 is employed in cell-based assays to study its role in neuronal death, axonal degeneration, and Schwann cell proliferation. Recombinant DR6 has been used as a tool to investigate its signaling pathways and downstream effects in various cell types.

• Immunological Research: DR6 is highly expressed in lymphoid tissues and is involved in the activation and differentiation of T and B lymphocytes. Recombinant DR6 is used to study immune cell regulation and death receptor signaling.

• Cancer Biology and Therapeutic Development: Recombinant DR6 has been utilized in studies targeting the DR6/APP axis to inhibit hematogenous metastasis. For example, peptidomimetic inhibitors of DR6/APP interaction have been developed and validated using recombinant DR6 in both in vitro and in vivo models of metastatic cancer.

• Western Blot (WB) and SDS-PAGE: Recombinant DR6 is commonly used as a positive control and immunogen in Western blotting and SDS-PAGE to validate antibody specificity and protein detection.

• Bioactivity Assays: Recombinant DR6 is validated for use in bioactivity assays to assess its functional properties and interactions with ligands or inhibitors.

Additional Context:

• Structural Biology: Recombinant DR6 has facilitated crystallographic studies, enabling the determination of its structure in complex with APP and providing insights into its activation mechanism.

• Neurodegeneration Models: DR6 has been studied in models of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, where recombinant DR6 is used to probe its role in motor neuron death and disease progression.

• General Research Use: Recombinant DR6 is widely used as a research reagent in various experimental protocols, including as a purified protein for in vitro assays and as an immunogen for antibody production.

Summary Table:

In summary, recombinant human DR6 is a versatile tool validated for protein interaction studies, cell biology, immunology, cancer research, and various biochemical assays in published research.

To reconstitute and prepare Recombinant Human DR6 protein for cell culture experiments, follow these best-practice steps:

• Check the datasheet or Certificate of Analysis (CoA) for your specific protein lot, as optimal conditions may vary by manufacturer and construct. If unavailable, the following general protocol is widely accepted for DR6 and similar recombinant proteins:

Reconstitution Protocol

1. Equilibrate the vial
   Allow the lyophilized protein vial to reach room temperature before opening to prevent condensation.

2. Centrifuge briefly
   Spin the vial in a microcentrifuge for 20–30 seconds to collect all powder at the bottom.

3. Add reconstitution buffer

   • For DR6, a common recommendation is to use sterile PBS (phosphate-buffered saline, pH 7.2–7.4) or sterile water.
   • A typical concentration is 100 μg/mL (e.g., add 100 μL buffer to 100 μg protein).
   • If using water, ensure subsequent dilution into buffered solutions before cell culture.

4. Dissolve gently

   • Add the buffer slowly along the vial wall to avoid foaming.
   • Gently swirl or invert; do not vortex as this may denature the protein.

5. Incubate
   Let the vial sit at room temperature for 15–30 minutes to ensure complete dissolution.

6. Check clarity
   The solution should be clear. If not, gently pipette up and down to aid dissolution.

Preparation for Cell Culture

• Dilute to working concentration
  Use cell culture medium or buffer containing a carrier protein such as 0.1% BSA, 5% HSA, or 10% FBS to stabilize the protein and prevent adsorption to plastic.
• Avoid repeated freeze-thaw cycles
  Aliquot the reconstituted protein and store at –20°C to –70°C for long-term use, or at 2–8°C for short-term (up to 1 month).
• Do not use water alone for final dilution
  Always dilute into buffered solutions or medium with carrier protein for cell culture applications to maintain protein stability and activity.

Storage

• Lyophilized protein: Store at –20°C to –80°C, desiccated.
• Reconstituted protein: Store aliquots at –20°C to –70°C for long-term, or 2–8°C for up to 1 week.

Additional Notes

• Always use sterile technique to avoid contamination.
• If the protein is fused to Fc or other tags, confirm compatibility with your assay or cell type.
• For functional assays, pre-test the protein in a pilot experiment to optimize concentration and incubation time.

Summary Table:

These steps will help ensure maximum activity and stability of recombinant DR6 protein for your cell culture experiments.