Recombinant Human TRAIL/Apo2L

Recombinant Human TRAIL/Apo2L is widely used in research because it is a potent, selective inducer of apoptosis in cancer cells while sparing most normal cells, making it a valuable tool for studying cell death mechanisms, cancer biology, and potential therapeutic strategies.

Key reasons to use recombinant human TRAIL/Apo2L in research applications:

• Selective Induction of Apoptosis in Tumor Cells: TRAIL/Apo2L binds to death receptors DR4 and DR5 on the cell surface, triggering apoptosis predominantly in transformed or cancerous cells, with minimal cytotoxicity to normal cells. This selectivity is crucial for studying tumor-specific cell death pathways and for preclinical evaluation of anti-cancer agents.

• Mechanistic Studies of Apoptosis: TRAIL activates both the extrinsic (caspase-8 mediated) and intrinsic (mitochondrial, caspase-9 mediated) apoptotic pathways, making it a robust model for dissecting apoptotic signaling, resistance mechanisms, and cross-talk with other cell death or survival pathways.

• Preclinical Cancer Therapy Research: Recombinant TRAIL/Apo2L has shown efficacy in reducing tumor burden and prolonging survival in animal models of various cancers, including breast, colon, and multiple myeloma. It is also used to evaluate combination therapies, as it can sensitize tumor cells to chemotherapeutic agents and irradiation.

• Immunology and Cell Biology: TRAIL is involved in immune cell-mediated cytotoxicity (e.g., by NK cells, monocytes, cytotoxic T cells), making it relevant for studies on immune surveillance, tumor-immune interactions, and immunotherapy.

• Clinical Relevance: Recombinant human TRAIL (e.g., dulanermin) is the only dual pro-apoptotic receptor agonist that directly activates both DR4 and DR5, and has advanced to clinical trials, underscoring its translational significance.

• Safety Profile: In preclinical studies, non-tagged, soluble, native-sequence TRAIL does not cause hepatotoxicity in animal models or normal human hepatocytes, supporting its use in translational research.

• Overcoming Drug Resistance: TRAIL/Apo2L can induce apoptosis in both drug-sensitive and drug-resistant cancer cell lines, making it a valuable tool for studying and overcoming therapeutic resistance.

In summary, recombinant human TRAIL/Apo2L is a versatile reagent for investigating apoptosis, cancer biology, drug resistance, and immune cell function, with strong relevance to both basic research and translational cancer therapy development.

Recombinant Human TRAIL/Apo2L can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with the assay system and the antibodies used. Many commercial ELISA kits for TRAIL/Apo2L are validated to detect both natural and recombinant forms, and recombinant TRAIL is routinely used as a standard in these assays.

Key considerations:

• Assay Compatibility: Confirm that your ELISA kit or custom assay is validated to detect recombinant TRAIL/Apo2L. Most sandwich ELISAs for TRAIL are designed to recognize both natural and recombinant proteins, but antibody specificity should be checked in the kit documentation or by consulting the antibody datasheets.
• Standard Preparation: Prepare serial dilutions of recombinant TRAIL/Apo2L in the same buffer or matrix as your samples to generate a standard curve. This allows accurate quantification by interpolation from the curve.
• Matrix Effects: Serum or plasma samples may contain interfering substances that affect quantification. Optimized sample diluents or buffer conditions may be required to minimize interference and ensure accuracy, especially when working with complex biological matrices.
• Protein Form: Ensure the recombinant TRAIL/Apo2L standard matches the form detected by your assay (e.g., soluble, extracellular domain, trimeric structure). Most ELISA kits are designed for soluble TRAIL, which is the typical recombinant form.
• Purity and Stability: Use highly purified recombinant protein and avoid repeated freeze/thaw cycles to maintain integrity and activity.

Protocol summary for using recombinant TRAIL/Apo2L as an ELISA standard:

1. Reconstitute or dilute recombinant TRAIL/Apo2L in assay buffer.
2. Prepare a series of known concentrations (e.g., 0, 100, 250, 500, 1000, 2000 pg/mL).
3. Run these standards in parallel with your samples.
4. Plot the standard curve (OD vs. concentration) and use curve-fitting software (e.g., 4PL or 5PL) for quantification.
5. Ensure sample and standard matrices are matched as closely as possible.

Summary:
You can use recombinant human TRAIL/Apo2L as a standard for ELISA quantification if your assay is validated for recombinant protein detection and you follow best practices for standard curve preparation and matrix matching. Always consult your specific ELISA protocol and antibody datasheets for compatibility.

Recombinant Human TRAIL/Apo2L has been extensively validated in published research for several key applications, primarily in the context of cancer biology and apoptosis studies.

Validated Applications in Published Research:

• Induction of Apoptosis in Cancer Cells:
  TRAIL/Apo2L is widely used to selectively induce apoptosis in a variety of human tumor cell lines, including breast, colon, prostate, multiple myeloma, and hepatocellular carcinoma cells. Its mechanism involves binding to death receptors DR4 and DR5, activating the extrinsic apoptotic pathway via caspase signaling.

• Preclinical Cancer Therapy Models:
  Systemic administration of recombinant TRAIL/Apo2L has been shown to be effective in killing human tumor xenografts in mice and prolonging survival in tumor-bearing animal models. It has also been evaluated in nonclinical toxicology studies for safety and efficacy prior to human trials.

• Combination Therapy Studies:
  TRAIL/Apo2L has been validated in combination with chemotherapeutic agents, irradiation, and targeted therapies to enhance tumor cell killing and overcome resistance mechanisms. For example, co-administration with DR5 agonist antibodies or agents that upregulate death receptors can synergistically increase apoptosis.

• Clinical Trials:
  Recombinant human Apo2L/TRAIL has been tested in Phase I and Ib clinical trials as a targeted therapy for solid tumors and hematologic malignancies, both as a monotherapy and in combination with other anticancer agents.

• Cell Culture Bioassays:
  TRAIL/Apo2L is routinely used in cell-based assays to study apoptosis, cytotoxicity, and signaling pathways in whole cells, including investigations into mechanisms of resistance and sensitization.

• Immunology and Mechanistic Studies:
  TRAIL/Apo2L has been used to study immune cell-mediated cytotoxicity, such as NK cell, monocyte, and cytotoxic T cell activity, as well as its role in cytokine production and cell metabolism.

Additional Notes:

• Most normal human cells are resistant to TRAIL-induced apoptosis, which underpins its selectivity for cancer cells and its therapeutic potential.
• Modified forms of TRAIL (e.g., Fc-fusion proteins) have been validated for improved bioavailability and activity in both in vitro and in vivo models.
• Safety assessments have included evaluation of hepatotoxicity and immune-mediated adverse effects in animal models and clinical studies.

Summary Table of Key Applications

These applications are supported by a broad body of peer-reviewed literature, confirming the utility of recombinant human TRAIL/Apo2L in both basic research and translational oncology.

To properly reconstitute and prepare Recombinant Human TRAIL/Apo2L protein for cell culture experiments, follow these general best practices based on manufacturer guidelines and scientific protocols:

1. Reconstitution

• Storage: Keep the lyophilized protein at –20°C or –80°C until ready to use.
• Reconstitution Solution: Use sterile, distilled water or the buffer specified in the product datasheet or Certificate of Analysis (CoA). Most TRAIL proteins are reconstituted in PBS or water.
• Procedure:
  • Allow the vial to warm to room temperature before opening to prevent condensation.
  • Carefully remove the cap.
  • Add the recommended volume of sterile water or buffer slowly along the inner wall of the vial to avoid foaming.
  • Do not vortex. Gently pipette up and down or swirl gently to dissolve the protein.
  • Let the solution sit at room temperature for 15–30 minutes to ensure complete dissolution.
  • The solution should be clear when fully dissolved.

2. Handling and Storage

• Immediate Use: Use the reconstituted protein as soon as possible for optimal activity.
• Short-Term Storage: Store the reconstituted protein at 2–8°C for up to 1 week (check specific product datasheet for exact duration).
• Long-Term Storage: For longer storage, aliquot the protein into single-use volumes and freeze at –20°C or –80°C. Avoid repeated freeze-thaw cycles.

3. Preparation for Cell Culture

• Dilution: Dilute the reconstituted TRAIL protein in cell culture medium or an appropriate buffer to the desired working concentration.
• Sterility: Ensure all steps are performed under sterile conditions to prevent contamination.
• Concentration: Typical working concentrations for TRAIL in cell culture range from 10–100 ng/mL, but optimize based on your cell type and experimental needs.

4. Additional Tips

• Wear gloves to prevent contamination by proteases.
• Avoid vigorous shaking or vortexing, as this can denature the protein and reduce biological activity.
• Always refer to the specific product datasheet for any unique instructions or recommendations.

By following these steps, you can ensure the proper reconstitution and preparation of Recombinant Human TRAIL/Apo2L protein for effective use in cell culture experiments.