Recombinant Human IL-1 RAcP

Recombinant Human IL-1 RAcP (Interleukin-1 Receptor Accessory Protein) is a critical component in IL-1 receptor signaling and is widely used in research to dissect IL-1-mediated pathways, study inflammation, and develop therapeutic strategies targeting cytokine signaling.

Key scientific reasons to use Recombinant Human IL-1 RAcP in research applications:

• Essential for IL-1 Signaling: IL-1 RAcP is required for the formation of the functional IL-1 receptor complex, enabling signal transduction upon IL-1 binding. Without RAcP, IL-1 cannot exert its physiological effects, making it indispensable for studies on IL-1 biology.
• Modulation of Inflammatory Responses: Recombinant IL-1 RAcP can be used to inhibit IL-1α-induced IL-8 secretion in cell models, demonstrating its utility in modulating inflammatory cytokine production and downstream effects.
• Decoy and Neutralization Studies: Soluble forms of IL-1 RAcP, especially when combined with soluble IL-1RII, dramatically increase the affinity for IL-1 and can neutralize IL-1 activity, allowing researchers to study mechanisms of cytokine inhibition and decoy receptor function.
• Structural and Mechanistic Investigations: Recombinant protein enables detailed biochemical and structural studies, such as mapping critical residues (e.g., Arg286) involved in receptor function or antibody binding, which is valuable for drug development and mechanistic insights.
• Cross-Species and Isoform Analysis: Recombinant human IL-1 RAcP shares high sequence identity with rodent orthologs, facilitating comparative studies and translational research using animal models.
• Therapeutic Target Validation: IL-1 RAcP is a novel target for blocking IL-1 function in human disease, and recombinant protein is essential for validating therapeutic antibodies or small molecules in preclinical assays.

Typical research applications include:

• Cell signaling assays to dissect IL-1 pathway activation.
• In vitro and in vivo models of inflammation, autoimmunity, and cytokine storm.
• Screening and validation of IL-1 pathway inhibitors.
• Structural biology and protein engineering for improved therapeutics.
• Decoy receptor studies to neutralize IL-1 activity.

Best practices:
Use recombinant IL-1 RAcP at concentrations validated for your cell type and assay system. Confirm purity and bioactivity using SDS-PAGE and functional assays. For mechanistic studies, consider pairing with IL-1R1 or IL-1RII to reconstitute the full receptor complex.

In summary:
Recombinant Human IL-1 RAcP is a versatile tool for investigating IL-1 signaling, modulating inflammatory responses, and validating therapeutic strategies targeting cytokine pathways.

Recombinant Human IL-1 RAcP can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and matched to the assay design. This approach is commonly used for quantitative ELISA, where recombinant proteins serve as standards to generate calibration curves for accurate measurement of analyte concentrations.

Essential context and best practices:

• Standard Curve Requirement: Quantitative ELISA assays require a standard curve generated from known concentrations of the target protein. Recombinant Human IL-1 RAcP is suitable for this purpose if its concentration and purity are well defined.
• Formulation Considerations: Recombinant proteins formulated with BSA (carrier protein) are generally recommended for use as ELISA standards due to enhanced stability. Carrier-free formulations may be preferred if BSA could interfere with your assay.
• Assay Compatibility: Ensure the recombinant IL-1 RAcP standard matches the isoform and epitope recognized by your ELISA antibodies. Most commercial ELISA kits for IL-1 RAcP include a recombinant standard for calibration, indicating compatibility.
• Reconstitution and Handling: Follow manufacturer or protocol guidelines for reconstitution (e.g., in PBS at specified concentrations) and storage to maintain protein integrity.
• Validation: It is critical to validate the standard in your specific assay context. Confirm linearity, sensitivity, and reproducibility of the standard curve using the recombinant protein.

Additional relevant information:

• Traceability: For rigorous quantification, standards should be traceable to reference materials or characterized by methods such as amino acid analysis or mass spectrometry, especially for clinical or regulatory applications.
• Documentation: Record lot numbers, concentration, and preparation details for reproducibility and data integrity.
• Limitations: Recombinant standards may differ from native proteins in post-translational modifications or folding, which can affect antibody recognition in some assays. Always verify assay performance with your chosen standard.

In summary, recombinant Human IL-1 RAcP is appropriate as an ELISA standard for quantification or calibration, provided it is compatible with your assay and properly validated.

Recombinant Human IL-1 RAcP has been validated in published research for several key applications, primarily in functional cell-based assays, protein-protein interaction studies, and as a tool for mechanistic investigation of IL-1 signaling pathways.

Validated Applications in Published Research:

• Functional Cell-Based Assays:
  Recombinant Human IL-1 RAcP has been used to inhibit IL-1α-induced IL-8 secretion in HepG2 human hepatocellular carcinoma cells, demonstrating its utility in studying cytokine signaling and inflammatory responses. The effective dose (ED₅₀) for this inhibition is reported as 0.8–4 μg/mL in the presence of recombinant IL-1R2 and IL-1α. This validates its use in cellular functional assays to dissect IL-1-mediated pathways.

• Protein-Protein Interaction Studies:
  The protein has been employed in protein pull-down assays to study interactions between IL-1RAcP and its binding partners, including the development and validation of inhibitory peptides targeting specific residues (e.g., Arg286) of IL-1RAcP. These studies are crucial for mapping interaction domains and for inhibitor screening.

• Mechanistic Studies of IL-1 Signaling:
  Recombinant IL-1 RAcP is essential for reconstituting the IL-1 receptor complex in vitro, allowing researchers to analyze the formation and function of the ternary signaling complex (IL-1β/IL-1R1/IL-1RAcP) and its downstream effects, such as induction of pro-inflammatory mediators (e.g., IL-6, IL-8). It is also used to study the role of IL-1RAcP in the assembly of other receptor complexes, such as the IL-33 receptor on mast cells and Th2 cells.

• Blocking/Neutralization Assays:
  The recombinant protein, especially in its soluble form, has been used to neutralize IL-1 activity by increasing the binding affinity of decoy receptors (e.g., IL-1RII), thereby serving as a tool in blocking assays to study cytokine inhibition and receptor decoy mechanisms.

• Development of Therapeutic Inhibitors:
  Studies have used recombinant IL-1RAcP to validate the efficacy of monoclonal antibodies and peptide inhibitors targeting specific regions of the protein, supporting its application in therapeutic development and validation.

Summary Table:

Additional Notes:

• While some commercial sources mention use in ELISA, Western blot, and immunohistochemistry, these applications are typically validated for antibodies rather than the recombinant protein itself. The primary published research applications for the recombinant protein are functional and mechanistic assays.
• Recombinant IL-1RAcP is also used as a standard or competitor in biochemical assays to study ligand binding and receptor complex formation.

If you require details on a specific application or protocol, please specify the context or experimental system.

To reconstitute and prepare Recombinant Human IL-1 RAcP protein for cell culture experiments, follow these steps:

• Centrifuge the vial briefly (20–30 seconds) before opening to ensure all lyophilized protein is at the bottom.
• Warm the vial to room temperature before opening to avoid condensation.
• Reconstitute the protein in sterile phosphate-buffered saline (PBS) or sterile distilled water. The recommended concentration is typically 200 μg/mL in PBS for carrier-free formulations, or 0.1–0.5 mg/mL in sterile distilled water for other preparations. Always check the product datasheet for specific instructions.
• Gently mix by pipetting up and down; do not vortex or shake vigorously, as this can denature the protein.
• Allow the protein to dissolve completely. If solubility issues arise, incubate the solution at 4 °C overnight.
• For long-term storage, aliquot the reconstituted protein and store at –20 °C to –70 °C. For short-term use, store at 2–8 °C for up to 1 week. Avoid repeated freeze-thaw cycles.
• For cell culture applications, consider adding a carrier protein (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) to stabilize the protein, especially if storing aliquots.
• Confirm protein concentration and integrity by SDS-PAGE if needed.

Summary of key steps:

• Centrifuge vial before opening.
• Reconstitute at 200 μg/mL in PBS or 0.1–0.5 mg/mL in sterile water.
• Mix gently, do not vortex.
• Aliquot and store at –20 °C to –70 °C for long-term, 2–8 °C for short-term.
• Add carrier protein for stability if required.

Application notes:

• For cell culture, use sterile technique throughout.
• Typical working concentrations for functional assays range from 0.8–4 μg/mL in the presence of IL-1 R2 and IL-1α.
• Always consult the specific product datasheet for any formulation-specific instructions.

If your protein is supplied as a lyophilized powder, these protocols are broadly applicable. Adjust buffer and concentration according to your experimental needs and the manufacturer’s recommendations.