Recombinant Human TRAIL R1

Recombinant Human TRAIL R1 is used in research applications primarily to study and manipulate apoptosis signaling pathways, especially in the context of cancer biology, due to its role as a death receptor that mediates selective tumor cell death.

Key scientific reasons to use recombinant TRAIL R1 include:

• Modeling Apoptosis Pathways: TRAIL R1 (also known as DR4 or TNFRSF10A) is a principal receptor for TRAIL (TNF-related apoptosis-inducing ligand), triggering the extrinsic apoptotic pathway upon ligand binding. This allows researchers to dissect the molecular mechanisms of apoptosis in various cell types, particularly tumor cells.

• Cancer Therapeutics Research: Activation of TRAIL R1 induces apoptosis selectively in cancer cells while sparing most normal cells, making it a valuable target for developing and testing anticancer therapies. Recombinant TRAIL R1 can be used to neutralize TRAIL activity or to study receptor-ligand interactions, resistance mechanisms, and combination strategies with chemotherapeutic agents.

• Functional Studies and Screening: Recombinant TRAIL R1 enables functional assays, such as:

  • Assessing cell sensitivity to TRAIL-induced apoptosis.
  • Screening for agonists or antagonists of TRAIL R1.
  • Investigating downstream signaling events, including caspase activation and PARP cleavage.

• Mechanistic Insights: Recent gene-editing studies show that TRAIL-R1 is a master regulator of apoptosis in certain carcinoma cell lines and can contribute to apoptosis during unresolved ER stress, highlighting its unique and non-redundant roles compared to TRAIL-R2.

• Immunology and Cell Migration: TRAIL R1 is expressed on hemopoietic cells and can influence immune cell activities, such as monocyte migration, providing additional research avenues in immunology and inflammation.

• Tool for Neutralization and Binding Studies: Recombinant TRAIL R1 can be used as a decoy receptor to neutralize TRAIL, preventing apoptosis in experimental systems, or as a binding partner in biochemical assays to characterize ligand specificity and affinity.

In summary, recombinant human TRAIL R1 is a versatile reagent for investigating apoptosis, cancer biology, immune cell function, and therapeutic development, offering a controlled system to probe TRAIL-mediated signaling and its modulation in health and disease.

You can use recombinant human TRAIL R1 as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it is compatible with your assay format. Many commercial ELISA kits for TRAIL R1 quantification specifically state that they recognize both natural and recombinant human TRAIL R1/DR4, and their protocols often use recombinant protein as the standard.

Key considerations:

• Formulation: Recombinant TRAIL R1 is often supplied lyophilized with a carrier protein such as BSA, which is suitable for use as an ELISA standard.
• Reconstitution: Follow the manufacturer’s instructions for reconstitution, typically in PBS with 0.1% BSA or similar stabilizer, to ensure protein stability and accurate concentration.
• Standard Curve Preparation: Serial dilutions of the recombinant standard should be prepared directly in the assay buffer or plate, as described in most ELISA kit protocols.
• Recognition by Antibodies: Ensure that the capture and detection antibodies in your ELISA recognize the recombinant form of TRAIL R1 you are using. Most sandwich ELISAs are validated to detect both natural and recombinant forms, but this should be confirmed for your specific assay.
• Calibration: Use the recombinant standard to generate a standard curve, typically analyzed using a 4- or 5-parameter logistic (4-PL or 5-PL) fit for best accuracy.

Limitations and best practices:

• The recombinant standard should match the native protein as closely as possible in terms of sequence and post-translational modifications, as differences may affect antibody recognition and quantification accuracy.
• If your recombinant TRAIL R1 is a fusion protein (e.g., Fc chimera), confirm that the ELISA antibodies target epitopes present in your standard.
• Always include a positive control of known concentration within the linear range of your standard curve to validate assay performance.

In summary, recombinant human TRAIL R1 is widely used as a standard in ELISA assays, but you must ensure compatibility with your assay system and follow best practices for preparation and calibration.

Recombinant Human TRAIL R1 (TNFRSF10A) has been validated for several key applications in published research, primarily centered around its role in apoptosis signaling, cancer therapy, and immune modulation. The main applications include:

1. Apoptosis Induction and Cell Death Studies
   Recombinant TRAIL R1 is used to study the mechanisms of TRAIL-induced apoptosis in cancer cells. It engages with TRAIL ligand to trigger the formation of the death-inducing signaling complex (DISC), leading to caspase activation and apoptosis. This is particularly relevant for understanding selective tumor cell death and resistance mechanisms.

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2. Cancer Immunotherapy and CAR-T Cell Development
   TRAIL R1 is a target for chimeric antigen receptor (CAR) T cell therapy. Recombinant TRAIL R1 has been used to validate the efficacy of TRAIL-R1-targeted CAR-T cells, which exhibit dual antitumor activity by inducing both TRAIL-R1–mediated apoptosis and CAR signal–induced cytolysis.

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3. Receptor-Ligand Binding and Affinity Studies
   The recombinant protein is used in binding assays to measure the interaction between TRAIL R1 and TRAIL ligand, often using techniques like biolayer interferometry (BLI) to determine affinity constants.

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4. Gene Editing and Functional Studies
   TRAIL R1 is used in gene-editing experiments (e.g., TALEN or CRISPR) to generate receptor-deficient cell lines, allowing researchers to dissect the specific contributions of TRAIL R1 versus TRAIL R2 in apoptosis and non-apoptotic signaling pathways.

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5. ELISA and Immunoassay Standards
   Recombinant TRAIL R1 is used as a standard in ELISA and other immunoassays to quantify receptor expression or ligand binding in cell or tissue samples.

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6. Immune Cell Function and Migration Studies
   TRAIL R1 expression on hematopoietic cells has been linked to immune cell activities such as monocyte migration, making it relevant for studies on immune surveillance and cell trafficking.

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7. Therapeutic Development and Drug Screening
   The protein is used in the development and screening of TRAIL-based therapeutics, including agonist antibodies and modified TRAIL ligands, to improve selectivity and efficacy against cancer cells.

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These applications highlight the utility of recombinant Human TRAIL R1 in both basic research and translational studies focused on cancer biology and immunotherapy.

To reconstitute Recombinant Human TRAIL R1 protein for cell culture experiments, dissolve the lyophilized protein at a concentration of 100 μg/mL in sterile PBS. If the formulation contains BSA as a carrier, ensure the PBS includes at least 0.1% human or bovine serum albumin to stabilize the protein. For carrier-free formulations, use sterile PBS alone.

Essential steps and best practices:

• Aseptic technique: Always use sterile equipment and solutions to prevent contamination.
• Gentle mixing: After adding the diluent, allow the vial to reconstitute for 15–30 minutes at room temperature with gentle agitation. Avoid vigorous shaking to prevent foaming and protein denaturation.
• Aliquoting: Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Storage: Store reconstituted protein at −20 °C or −80 °C in a manual defrost freezer. Avoid repeated freeze-thaw cycles.
• Dilution for cell culture: Prior to use, dilute the stock solution to the desired working concentration using cell culture medium or PBS. If using directly in cell culture, ensure the final buffer composition is compatible with your cells (e.g., isotonicity, absence of toxic additives).

Additional considerations:

• If the protein is supplied without carrier protein, adding 0.1% BSA or HSA during reconstitution can help prevent adsorption to tube walls and loss of activity, especially at low concentrations.
• Briefly centrifuge the vial before opening to collect all lyophilized material at the bottom.
• For proteins prone to aggregation, gentle pipetting or slow inversion is preferable to vortexing.
• Always consult the specific product datasheet for any unique requirements or recommended buffers.

Summary protocol:

1. Briefly centrifuge the vial.2. Add sterile PBS (with 0.1% BSA if recommended) to achieve 100 μg/mL.3. Mix gently and allow to dissolve for 15–30 minutes at room temperature.4. Aliquot and store at −20 °C or −80 °C.5. Dilute to working concentration in cell culture medium immediately before use.

This protocol ensures optimal solubility, stability, and biological activity of recombinant TRAIL R1 for cell-based assays.