Recombinant Rat IL-10 (Cys 167 Tyr)

Recombinant Rat IL-10 (Cys 167 Tyr) is used in research applications because it is a biologically active, anti-inflammatory cytokine that plays a critical role in immunoregulation, inflammation control, and tissue protection, with the Cys167Tyr mutation specifically enhancing its stability and activity in recombinant expression systems.

Key reasons to use this protein in your research include:

• Immunoregulatory and Anti-inflammatory Functions: IL-10 suppresses the synthesis of pro-inflammatory cytokines, enhances B cell survival and proliferation, and increases antibody production. It is essential for maintaining immune homeostasis and preventing excessive inflammatory responses.
• Enhanced Biological Activity and Stability: The Cys167Tyr (or Cys149Tyr, depending on numbering) mutation replaces a cysteine residue with tyrosine, which improves the protein’s ability to fold correctly and remain active after recombinant expression and refolding. Native-sequence rat IL-10 often shows reduced or absent activity, whereas the Cys167Tyr variant is reliably bioactive and easier to produce in bacterial systems.
• Modeling Disease and Therapeutic Studies: Recombinant rat IL-10 is widely used in models of inflammation, autoimmunity, and cancer. It has been shown to protect tissues (e.g., lung alveolar cells in neonatal rats), promote tolerance to self and foreign antigens, and enhance antitumor immune responses when used as an adjuvant in immunotherapy studies.
• Mechanistic Studies: IL-10 is involved in key signaling pathways such as JAK-STAT and can block NF-κB activity, making it valuable for dissecting immune signaling mechanisms.
• Cross-Species Activity: While human IL-10 is active on mouse cells, mouse and rat IL-10 are not active on human cells, so using recombinant rat IL-10 is necessary for rat-specific or cross-species studies.

Typical applications include:

• In vitro cell culture assays to study immune cell regulation and cytokine signaling.
• In vivo models of inflammation, autoimmunity, or tissue injury to assess protective or therapeutic effects.
• As a standard or control in ELISA and other immunoassays.

Summary: Use recombinant rat IL-10 (Cys 167 Tyr) when you require a robust, biologically active IL-10 for rat models or in vitro studies, especially where reliable folding, stability, and activity are critical for experimental reproducibility and interpretation.

You can use recombinant rat IL-10 (Cys167Tyr) as a standard for quantification or calibration in ELISA assays, provided that the ELISA kit is validated to recognize recombinant IL-10 and the mutation (Cys167Tyr) does not significantly alter the antigenic epitopes recognized by the kit antibodies or the biological activity relevant to your assay.

Supporting details:

• Most commercial rat IL-10 ELISA kits are designed to detect both natural and recombinant rat IL-10, and their standards are often recombinant proteins expressed in E. coli.
• The standard curve in these kits is typically generated using recombinant IL-10, and dose-response curves for natural and recombinant IL-10 are reported to be parallel, indicating comparable recognition and quantification.
• The Cys167Tyr mutation is not a common natural variant, so you must ensure that this substitution does not affect antibody binding in your specific ELISA. If the antibodies target regions distant from residue 167, or if the mutation does not disrupt the protein’s conformation, the recombinant mutant should be suitable. However, if the kit documentation or validation data do not explicitly mention compatibility with this variant, you should perform a parallelism test: compare the standard curve generated with your recombinant mutant to that of the kit’s standard to confirm equivalence.
• For absolute quantification, any difference in immunoreactivity or biological activity due to the mutation could affect accuracy. For relative quantification or calibration, as long as the mutant is recognized equivalently, it is acceptable.

Best practices:

• Validate the use of your recombinant IL-10 (Cys167Tyr) by running a standard curve alongside the kit’s standard and assessing parallelism and recovery.
• Confirm that the ELISA kit’s antibodies do not specifically require the wild-type cysteine at position 167 for binding.
• Use the recombinant protein only for research purposes, as all referenced kits are for research use only.

If you do not have validation data for the Cys167Tyr mutant, it is recommended to contact the kit provider for epitope information or perform your own validation experiments.

Recombinant Rat IL-10 (Cys 167 Tyr) has been validated in published research primarily for applications involving the modulation of immune responses, protection against lung injury, and as a tool for studying cytokine biology in vitro and in vivo.

Key validated applications include:

• Protection against lung injury in neonatal rats: Recombinant rat IL-10 (Cys 167 Tyr) has been used in both in vitro and in vivo models to demonstrate protective effects on alveolar type II cells and lung tissue. Specifically, intratracheal administration of rIL-10 prior to hyperoxic exposure in newborn rats reduced pulmonary inflammation, prevented loss of alveolar type II cells, and inhibited the release of proinflammatory mediators during early-stage hyperoxia. These studies validate its use in models of neonatal lung injury and inflammation.

• Immunomodulation and anti-inflammatory studies: This IL-10 variant has been used to investigate its ability to inhibit pro-inflammatory cytokine synthesis, enhance B cell survival and proliferation, and regulate immune responses, including peripheral T cell tolerance and suppression of NK cell activity. These applications are typically validated in cell-based assays and animal models.

• Functional cell-based assays: The biological activity of recombinant rat IL-10 (Cys 167 Tyr) has been validated in cell proliferation assays, such as those using MC/9-2 mouse mast cells, to confirm its cytokine function and potency.

• Tool for cytokine engineering and regenerative medicine research: Engineered forms of IL-10, including Cys 167 Tyr, are used in studies aiming to modulate immune responses for tissue repair and regeneration, although specific published validations for this exact variant in regenerative medicine are less frequently cited.

• General cytokine biology research: The Cys 167 Tyr mutation improves protein folding and stability, making this variant a preferred tool for investigating IL-10’s biological actions in rat models, especially where native-sequence rat IL-10 is less active or unstable.

Summary Table: Validated Applications

Additional Notes:

• The Cys 167 Tyr mutation (sometimes referred to as Tyr149 in older literature) is specifically engineered to improve recombinant protein yield and activity, making it a standard for rat IL-10 research.
• While the above applications are validated in published research, the protein is also widely used as a standard or control in immunological assays, such as ELISA and neutralization studies, though these are not always explicitly detailed in publications.

If you require protocols or more specific details on a particular application, please specify the experimental context.

To reconstitute and prepare Recombinant Rat IL-10 (Cys 167 Tyr) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of at least 100 µg/mL, then further dilute as needed for your assay. Use gentle mixing and avoid vortexing to prevent protein denaturation.

Step-by-step protocol:

• Reconstitution:

  • Add sterile, endotoxin-free water or buffer (e.g., 5 mM Tris, pH 7.2) to the lyophilized protein to achieve a concentration between 0.1–1.0 mg/mL for stock solutions.
  • For initial solubilization, a concentration of ≥100 µg/mL is recommended.
  • Gently pipette the solution down the sides of the vial to dissolve the protein. Do not vortex.
  • Allow the protein to fully dissolve at room temperature for several minutes.

• Aliquoting and Storage:

  • After reconstitution, aliquot the stock solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at 2–8°C for up to one month or at –20°C to –70°C for long-term storage.
  • For long-term storage, consider adding a carrier protein (e.g., 0.1% HSA or BSA) to stabilize the protein.
  • Avoid repeated freeze-thaw cycles to maintain bioactivity.

• Preparation for Cell Culture:

  • Prior to use, dilute the stock solution in cell culture medium or appropriate buffer to the desired working concentration (typically in the ng/mL range, depending on your experimental design).
  • Ensure the final solution is sterile and compatible with your cell culture system.

• Handling Notes:

  • Mix gently by inversion or slow pipetting; avoid foaming.
  • If using for bioassays, confirm biological activity (e.g., ED50 for MC/9 cell inhibition is <10 ng/mL).
  • Check endotoxin levels if required for sensitive cell types (should be <0.1 EU/µg).

Summary Table:

Best Practices:

• Always use aseptic technique during reconstitution and dilution.
• Validate protein activity in your specific cell culture system.
• Consult the certificate of analysis for specific lot instructions if available.

If you require a specific buffer for your cell type or application, adjust accordingly, but ensure the buffer is compatible with both the protein and your cells.