Recombinant Human IL-6Rα

Recombinant Human IL-6Rα is widely used in research to study and manipulate the interleukin-6 (IL-6) signaling pathway, which is central to immune regulation, inflammation, and various disease processes, including autoimmune disorders and cancer.

Key reasons to use recombinant human IL-6Rα in research applications:

• Mechanistic Studies of IL-6 Signaling: IL-6Rα is the specific receptor subunit that binds IL-6, initiating downstream signaling through the gp130 co-receptor. Using recombinant IL-6Rα allows precise dissection of ligand-receptor interactions, receptor binding affinities, and the molecular mechanisms underlying IL-6-mediated signal transduction.

• Development and Testing of Therapeutics: Recombinant IL-6Rα is essential for screening and characterizing inhibitors, antibodies, or small molecules that block IL-6/IL-6Rα interactions. Such studies are foundational for developing new anti-inflammatory or anti-cancer drugs, as IL-6Rα blockade has proven clinical efficacy in diseases like rheumatoid arthritis.

• Cellular Assays and Functional Studies: Adding recombinant IL-6Rα to cell cultures enables the study of IL-6-dependent cellular responses, such as STAT3 phosphorylation, cell proliferation, differentiation, and migration. This is particularly useful for modeling disease states or testing the effects of IL-6 pathway modulation in vitro.

• Structural and Biophysical Research: Recombinant IL-6Rα is used in binding assays (e.g., ELISA, SPR) and structural studies to map interaction sites, analyze mutant proteins, and design improved therapeutic agents or research tools.

• Modeling Pathological Conditions: Since dysregulated IL-6 signaling is implicated in autoimmune diseases, chronic inflammation, and cancer, recombinant IL-6Rα is valuable for creating in vitro and in vivo models to study disease mechanisms and evaluate potential interventions.

Additional context:

• IL-6Rα exists in both membrane-bound and soluble forms; recombinant proteins can model both, enabling studies of classic and trans-signaling pathways.
• Recombinant IL-6Rα is often used in combination with IL-6 or its inhibitors to dissect pathway specificity and therapeutic potential.

In summary, recombinant human IL-6Rα is a critical reagent for elucidating IL-6 biology, developing targeted therapies, and modeling diseases where IL-6 signaling plays a pivotal role.

Yes, recombinant human IL-6Rα can be used as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately determined. This approach is widely accepted in research ELISA protocols for quantifying soluble IL-6Rα in biological samples.

Supporting details:

• ELISA kits for human IL-6Rα routinely use recombinant human IL-6Rα as the standard. These standards are typically produced in eukaryotic expression systems (e.g., Sf21 insect cells) and are calibrated to provide accurate quantification of both recombinant and natural forms of IL-6Rα.
• Parallelism and dilution linearity: Validation studies show that standard curves generated with recombinant IL-6Rα are parallel to those obtained with natural IL-6Rα, indicating that the recombinant protein is suitable for calibration and quantification in ELISA assays.
• Preparation and use: The recombinant protein should be highly purified, and its concentration must be precisely known. Serial dilutions are prepared to generate a standard curve, which is then used to interpolate the concentration of IL-6Rα in unknown samples.
• Assay specificity: ELISA kits designed for IL-6Rα quantification are validated to recognize both natural and recombinant forms, ensuring accurate measurement across sample types.
• Best practices: Use the same buffer and diluent for both standards and samples to minimize matrix effects. Always verify that the recombinant standard is compatible with your specific ELISA kit and detection antibodies.

Limitations:

• The recombinant standard should be for research use only and not for diagnostic procedures unless specifically validated for clinical use.
• Ensure the recombinant protein matches the isoform and glycosylation status relevant to your assay, as differences may affect antibody recognition in some cases.

Summary Table: Recombinant IL-6Rα as ELISA Standard

In summary: Recombinant human IL-6Rα is scientifically appropriate as a standard for ELISA quantification and calibration, provided it is validated for your assay system and used according to best laboratory practices.

Recombinant Human IL-6Rα has been validated for several key applications in published research, primarily in studies of cytokine signaling, antibody screening, and functional cell-based assays.

Validated Applications:

• Binding and Bioactivity Assays: Recombinant human IL-6Rα is routinely used to measure its ability to bind recombinant human IL-6, confirming its bioactivity in in vitro systems. This is essential for characterizing IL-6/IL-6Rα interactions and downstream signaling.

• ELISA and Kinetics Assays: It serves as a target in enzyme-linked immunosorbent assays (ELISA) and kinetic binding studies, particularly for screening antibodies that block or modulate IL-6/IL-6Rα interactions. These assays are critical for therapeutic antibody development and mechanistic studies.

• Cell-based Functional Assays: Recombinant IL-6Rα is used in cell lines lacking endogenous IL-6Rα to reconstitute IL-6 signaling, enabling analysis of STAT3 phosphorylation and other downstream effects. This includes validation of antibody neutralization activity and investigation of trans-signaling mechanisms.

• Antibody Discovery and Hybridoma Generation: It is employed as an immunogen or fusion partner (e.g., in Hyper-IL-6 constructs) for generating monoclonal antibodies targeting the IL-6/IL-6Rα complex, facilitating the discovery of novel therapeutic candidates.

• Molecular Interaction Studies: Recombinant soluble IL-6Rα (seIL-6R) is used in molecular docking and structural studies to analyze its interaction with gp130, the signal-transducing component of the IL-6 receptor complex.

Additional Context:

• Therapeutic Validation: Recombinant human IL-6Rα is central to the development and validation of IL-6 receptor inhibitors, which are clinically used in autoimmune diseases such as rheumatoid arthritis. These studies often use recombinant proteins to model receptor blockade and assess therapeutic efficacy.

• Standardization and Quantification: It is used as a standard in quantification assays for IL-6 signaling, including screening and release assays for antibodies that block IL-6 signaling.

Summary Table:

These applications demonstrate the versatility of recombinant human IL-6Rα in both basic and translational research, particularly in immunology, cell signaling, and therapeutic antibody development.

To reconstitute and prepare Recombinant Human IL-6Rα protein for cell culture experiments, dissolve the lyophilized protein in sterile water to a concentration of 0.1 mg/mL, then dilute further in appropriate aqueous buffers as needed for your assay. For optimal stability and to prevent adsorption, add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA), especially if you plan to store aliquots for future use.

Detailed protocol:

• Reconstitution:

  • Allow the lyophilized IL-6Rα vial to equilibrate to room temperature before opening to minimize condensation.
  • Add sterile water directly to the vial to achieve a final concentration of 0.1 mg/mL (or as specified by your supplier’s datasheet).
  • Gently swirl or invert the vial to dissolve. Avoid vigorous vortexing, which can denature the protein.
  • If the protein is difficult to dissolve, let it sit at room temperature for 10–30 minutes, then gently mix again.

• Dilution for cell culture:

  • Prepare working solutions by diluting the reconstituted stock in sterile cell culture medium or PBS, ideally containing 0.1% BSA or HSA to stabilize the protein.
  • Filter-sterilize the final working solution if sterility is required and the protein is compatible with filtration.

• Aliquoting and storage:

  • Aliquot the reconstituted stock to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • Store aliquots at –20°C or colder for long-term storage. Avoid storing at 4°C for extended periods, as this can promote degradation.

• Usage in cell culture:

  • Thaw aliquots on ice before use.
  • Add the protein to cell culture at the desired final concentration, typically in the range of 1–100 ng/mL, depending on your experimental design and cell type.
  • Include appropriate controls (e.g., vehicle only, carrier protein only).

Additional notes:

• Always consult the specific product datasheet for any lot-specific instructions, as some recombinant proteins may require unique handling or reconstitution buffers.
• If using for ELISA or bioassays, reconstitute and dilute in the recommended assay diluent to ensure compatibility with the detection system.

Summary of best practices:

• Use sterile technique throughout.
• Add a carrier protein for stability.
• Avoid repeated freeze-thaw cycles.
• Follow any supplier-specific instructions for optimal results.

These steps will ensure that your recombinant human IL-6Rα protein is properly prepared for reliable and reproducible cell culture experiments.