Recombinant Human TNF RI

Recombinant Human TNF RI (Tumor Necrosis Factor Receptor I, also known as TNFRSF1A) is widely used in research applications to study and modulate TNF-α signaling, investigate inflammatory pathways, and develop therapeutic strategies targeting TNF-mediated diseases.

Key reasons to use recombinant human TNF RI in research include:

• Specific Inhibition of TNF-α Activity: Recombinant TNF RI acts as a decoy receptor, binding to TNF-α with high affinity and preventing it from interacting with cell surface receptors, thereby inhibiting downstream pro-inflammatory signaling. This is particularly useful for dissecting the role of TNF-α in inflammation, apoptosis, and immune regulation.

• Modeling and Modulation of Inflammatory Responses: Most biological effects of TNF-α, including those relevant to autoimmune diseases and cancer, are mediated via TNF RI. Using recombinant TNF RI allows researchers to selectively block or mimic TNF-α signaling, facilitating studies on cytokine networks, cell death, and survival pathways.

• Therapeutic Development and Mechanistic Studies: Soluble forms of TNF RI are used as models for therapeutic agents that neutralize TNF-α, such as those used in rheumatoid arthritis and other chronic inflammatory diseases. Recombinant TNF RI can be used in preclinical assays to evaluate the efficacy of TNF-targeted drugs or to screen for novel inhibitors.

• Bioassays and Quantification: Recombinant TNF RI is commonly used as a standard or reagent in bioassays, such as ELISA, to quantify TNF-α levels or to assess the neutralizing capacity of antibodies and small molecules.

• Resolution of Inflammation: The release of soluble TNF RI helps resolve inflammatory reactions by down-regulating cell surface TNF receptors and sequestering free TNF-α, making recombinant TNF RI a valuable tool for studying mechanisms of inflammation resolution.

• Signal Transduction Research: TNF RI is involved in key signaling pathways, including activation of NF-κB and regulation of apoptosis and angiogenesis. Recombinant forms enable detailed mechanistic studies of these pathways in vitro.

Additional considerations:

• Recombinant human TNF RI is available in various formats (e.g., Fc chimeras, soluble forms) to suit different experimental needs, such as increased stability or enhanced detection in assays.
• It is important to select the appropriate form and concentration for your specific application, as the biological activity and binding characteristics may vary.

In summary, recombinant human TNF RI is a versatile reagent for investigating TNF-α biology, modulating inflammatory responses, and supporting the development of anti-TNF therapeutics in a controlled and reproducible manner.

Yes, recombinant human TNF RI (soluble TNF receptor I) can be used as a standard for quantification or calibration in ELISA assays, provided it is appropriately validated for your specific assay format. This is a common practice in commercial ELISA kits and custom assay development.

Key considerations and supporting details:

• Commercial ELISA kits for human TNF RI routinely use recombinant human TNF RI as the standard for generating calibration curves. These standards are typically non-glycosylated, E. coli-expressed proteins representing the extracellular domain of the receptor, and are supplied with the kit for quantification of both recombinant and natural forms in biological samples.

• Parallelism and accuracy: Validation data from established kits show that the standard curves generated with recombinant TNF RI are parallel to those obtained with natural TNF RI in biological samples, indicating that the recombinant standard is suitable for accurate quantification. This parallelism is essential for reliable quantification across different sample types.

• Calibration and quantification: The recombinant standard is used to create a standard curve, against which unknown sample concentrations are interpolated. The accuracy of quantification depends on the standard being well-characterized and the antibodies in the assay recognizing both recombinant and native forms equivalently.

• Assay validation: For best practice, ensure that your ELISA is validated for:

  • Linearity and parallelism between the recombinant standard and endogenous analyte in your sample matrix.
  • Recovery and matrix effects to confirm that the recombinant standard behaves similarly to the native protein in your specific sample type.
  • Precision and accuracy within the assay’s working range.

• Standard preparation: Follow the manufacturer’s instructions for reconstitution and dilution of the recombinant standard to ensure consistency and reproducibility.

• Reference to international standards: When available, some kits calibrate their recombinant standards against NIBSC/WHO international standards for additional traceability.

Summary Table: Use of Recombinant Human TNF RI as ELISA Standard

In summary: You can use recombinant human TNF RI as a standard for ELISA quantification, as long as you validate that your assay detects both recombinant and native forms equivalently and that the standard curve is appropriate for your sample matrix. This approach is widely accepted in research and commercial assay development.

Recombinant Human TNF RI (Tumor Necrosis Factor Receptor I, also known as TNFRSF1A or p55 TNFR) has been validated in published research for several key applications:

• Bioassays: Used to assess biological activity, such as the inhibition of TNF-α-mediated cytotoxicity in cell-based assays (e.g., L929 mouse fibroblast cytotoxicity assay).
• ELISA (Enzyme-Linked Immunosorbent Assay): Employed as a standard or capture/detection reagent for quantifying TNF-α or soluble TNF RI in biological samples.
• Functional Assays: Used to study TNF-α signaling, receptor-ligand interactions, and to block or neutralize TNF-α activity in vitro.
• Western Blot: Applied as a positive control or to validate antibody specificity for TNF RI detection.
• Blocking Assays: Utilized to inhibit TNF-α activity in cell culture or biochemical systems, demonstrating the receptor’s antagonistic properties.
• Protein-Protein Interaction/Binding Assays: Used to characterize interactions between TNF RI and TNF-α or other binding partners.

Supporting details and examples from published research:

• Bioassay: Recombinant human TNF RI has been used to inhibit TNF-α-mediated cytotoxicity in L929 cells, a standard assay for TNF bioactivity.
• ELISA: Multiple studies have used recombinant TNF RI as a standard or reagent in ELISA to quantify TNF-α or soluble TNF RI in serum, plasma, or cell culture supernatants.
• Functional/Blocking Assays: Recombinant TNF RI is validated for blocking TNF-α activity, both in vitro and in animal models, to study inflammation and immune responses.
• Western Blot: Used as a control protein to confirm antibody specificity for TNF RI.
• Protein-Protein Interaction: Applied in binding assays to study the affinity and kinetics of TNF-α/TNF RI interactions.

Summary Table of Validated Applications

Additional notes:

• Recombinant TNF RI is often used as a soluble receptor to antagonize TNF-α in mechanistic studies of inflammation and immune regulation.
• Published research has validated its use in both human and animal model systems, particularly in studies of inflammatory diseases and cytokine signaling.

If you require details on a specific application protocol or a particular research context, please specify.

Reconstitution Protocol

Reconstituting lyophilized TNF RI protein requires careful attention to technique to maintain protein activity and stability. Begin by allowing both the vial and your reconstitution buffer to equilibrate to room temperature before opening. This equilibration step is critical for preventing osmotic stress on the protein.

Once at room temperature, briefly centrifuge the vial or gently tap it to ensure all lyophilized material collects at the bottom. This prevents the protein from remaining as a film on the vial walls, which can reduce reconstitution efficiency.

Reconstitution Buffer and Concentration

TNF RI protein is typically reconstituted at 100 μg/mL in sterile phosphate-buffered saline (PBS). For carrier-containing formulations, ensure your PBS contains at least 0.1% human or bovine serum albumin (BSA). The presence of BSA enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations. If using a carrier-free formulation, reconstitute in sterile PBS without additional carrier protein.

Reconstitution Procedure

Add the appropriate volume of buffer to achieve your target concentration. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation. This incubation period permits the protein to fully dissolve. Critically, avoid vigorous shaking or vortexing, as this causes foaming and protein denaturation. If the product exhibits flakes or particulates after initial mixing, continue gentle mixing for a couple of hours at room temperature, then overnight at 4°C.

Storage and Stability

After reconstitution, aliquot the protein into volumes greater than 20 μL to minimize surface area exposure. Store reconstituted aliquots at ≤ –20°C for long-term storage. Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these compromise protein integrity. For short-term use in cell culture, the protein may be stored at 2-8°C for up to one month.

Lyophilized protein stored desiccated at -20°C to -70°C remains stable for 6-12 months. Upon receipt of your protein, store it immediately at the recommended temperature to maintain stability.

Considerations for Cell Culture Applications

For cell or tissue culture applications, the carrier-containing formulation is preferred over carrier-free versions, as BSA does not typically interfere with most cell-based assays and provides superior protein stability. Ensure all reconstitution materials and buffers are sterile to prevent contamination of your cell cultures. If long-term storage beyond one month is required, maintain samples at -20°C or -70°C in appropriate aliquots rather than storing at refrigeration temperatures.