Using Recombinant Human IL-10 Rβ (IL-10 receptor beta) in research applications is essential for studying the molecular mechanisms of IL-10 signaling, dissecting receptor-ligand interactions, and developing or optimizing immunomodulatory therapies. IL-10 Rβ is a critical component of the functional IL-10 receptor complex, which mediates the anti-inflammatory and immunoregulatory effects of IL-10.

Key scientific reasons to use Recombinant Human IL-10 Rβ:

• Mechanistic Studies of IL-10 Signaling: IL-10 Rβ, together with IL-10 Rα, forms the high-affinity receptor complex required for IL-10-mediated signal transduction. Recombinant IL-10 Rβ enables in vitro reconstitution of the receptor complex, allowing detailed analysis of ligand binding, receptor stoichiometry, and downstream signaling events such as STAT1 and STAT3 activation.

• Protein Engineering and Affinity Studies: Recombinant IL-10 Rβ is crucial for screening and characterizing IL-10 variants with altered receptor affinity. Enhanced binding to IL-10 Rβ has been shown to increase receptor heterodimerization and potentiate IL-10’s immunomodulatory effects, which is valuable for therapeutic protein engineering and drug development.

• Functional Assays and Drug Screening: Recombinant IL-10 Rβ can be used in cell-based assays to evaluate the biological activity of IL-10 or its engineered variants, including their ability to suppress pro-inflammatory cytokine production, modulate antigen presentation, and regulate immune cell function.

• Structural and Biophysical Research: Purified recombinant IL-10 Rβ is required for structural studies (e.g., cryo-EM, X-ray crystallography) to elucidate the architecture of the IL-10/IL-10 receptor complex, providing insights into receptor activation and informing rational drug design.

• Therapeutic Development: Understanding and manipulating IL-10 Rβ interactions is relevant for developing novel immunotherapies, particularly for autoimmune diseases, inflammatory disorders, and cancer, where modulation of IL-10 signaling can have therapeutic benefits.

Summary of applications:

• Dissecting IL-10 receptor complex assembly and signaling.
• Engineering IL-10 variants with improved therapeutic properties.
• Developing and validating functional assays for immunomodulation.
• Structural characterization of cytokine-receptor interactions.
• Supporting preclinical and translational research in immunology and oncology.

In summary, recombinant human IL-10 Rβ is a fundamental reagent for advancing both basic and translational research on IL-10 biology and its therapeutic potential.

No, you should not use recombinant Human IL-10 Rβ (the receptor subunit) as a standard for quantification or calibration in your ELISA assays for human IL-10.

Here’s why:

• ELISA standards are typically recombinant forms of the analyte itself—in this case, recombinant human IL-10 cytokine (the ligand), not its receptor.
• IL-10 Rβ is a receptor subunit that binds IL-10 as part of the signaling complex, but it is not the target molecule measured by IL-10 ELISA kits.
• ELISA kits for IL-10 are designed to detect and quantify IL-10 protein using antibodies specific to IL-10. These antibodies will not recognize IL-10 Rβ, so using IL-10 Rβ as a standard will not generate a valid standard curve for IL-10 quantification.

For accurate quantification, always use a recombinant human IL-10 protein standard that is specifically validated for use in your ELISA kit. This ensures proper calibration and accurate measurement of IL-10 concentration in your samples.

Applications of Recombinant Human IL-10 Rβ in Published Research

Recombinant IL-10 Rβ has been validated for several key applications in published research, primarily centered on receptor characterization and signaling studies.

Biophysical Characterization

The primary application of recombinant IL-10 Rβ involves surface plasmon resonance (SPR) studies to characterize binding kinetics and affinity measurements. In published research, biotinylated IL-10 Rβ has been immobilized onto chip surfaces to quantify the binding interactions between IL-10 variants and the receptor, enabling precise determination of apparent binding affinities at nanomolar concentrations.

Receptor Complex Analysis

Recombinant IL-10 Rβ has been utilized to investigate receptor heterodimerization and complex stoichiometry. Researchers have employed quantitative imaging studies with IL-10 Rβ to examine how engineered IL-10 variants recruit the receptor into active cell surface signaling complexes, providing insights into the native IL-10/IL-10 receptor complex architecture.

Signaling and Functional Studies

The receptor has supported investigations of downstream signaling activation, particularly in measuring STAT1 and STAT3 phosphorylation in human primary immune cells including monocytes and CD8+ T cells. These applications have been critical for validating how enhanced IL-10 Rβ binding affinity translates into improved immunomodulatory activities and gene expression programs.

Therapeutic Development

Recombinant IL-10 Rβ has been instrumental in screening and validation assays for engineered IL-10 variants intended for therapeutic applications. These studies have demonstrated how receptor-binding optimization can enhance anti-inflammatory efficacy at subsaturating doses, which is particularly relevant for clinical therapeutic interventions.

To reconstitute and prepare Recombinant Human IL-10 Rβ protein for cell culture experiments, follow these best-practice steps based on current protocols for recombinant cytokine receptor proteins:

1. Equilibrate the vial: Allow the lyophilized protein to reach room temperature before opening to prevent condensation.

2. Centrifuge briefly: Spin the vial in a microcentrifuge for 20–30 seconds to collect all material at the bottom.

3. Reconstitution buffer: Use sterile, endotoxin-free PBS (pH 7.2–7.4) as the primary buffer. If the product datasheet specifies, you may also use sterile water, but PBS is generally preferred for receptor proteins.

4. Protein concentration: Reconstitute to a concentration between 0.1–1.0 mg/mL. A typical starting point is 0.1–0.2 mg/mL. Avoid exceeding 1 mg/mL to prevent solubility issues.

5. Carrier protein: To enhance stability and prevent adsorption to plastic, add 0.1%–1% endotoxin-free human or bovine serum albumin (HSA/BSA) to the buffer, especially if you plan to store aliquots or use low concentrations.

6. Mixing: Gently swirl or tap the vial to dissolve the protein. Do not vortex or shake vigorously, as this can denature the protein.

7. Incubation (if needed): If the protein does not dissolve immediately, allow it to incubate at 4°C for several hours or overnight.

8. Aliquot and storage: After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles. Store aliquots at –20°C to –70°C for long-term storage, or at 2–8°C for short-term use (up to 1 month).

9. Working dilution: For cell culture, dilute the reconstituted stock into your culture medium to the desired final concentration. Include carrier protein (e.g., 0.1% BSA) in all working solutions to maintain stability.

10. Sterility: Always use sterile technique throughout the process to prevent contamination.

Example protocol for IL-10 Rβ:

• Add sterile PBS (pH 7.2–7.4) with 0.1% BSA to the vial to achieve 0.2 mg/mL.
• Gently mix until fully dissolved.
• Aliquot and store at –20°C or colder.
• For cell culture, dilute to the required concentration in complete medium, ensuring the presence of carrier protein.

Additional notes:

• Avoid repeated freeze-thaw cycles, as this can denature the protein and reduce activity.
• If the protein forms a film or precipitate, ensure gentle mixing and, if necessary, brief centrifugation to collect the solution.
• Always consult the product-specific datasheet for any unique requirements.

These steps are consistent with standard protocols for recombinant cytokine receptors and ensure optimal protein stability and biological activity in cell culture applications.