Recombinant Rat IL-6

Recombinant Rat IL-6 is widely used in research applications due to its critical role as a multifunctional cytokine in regulating immune responses, inflammation, hematopoiesis, and disease modeling.

Key reasons to use recombinant rat IL-6 in research include:

• Modeling Inflammatory and Immune Responses: IL-6 is a central mediator in both acute and chronic inflammation, influencing B cell maturation, T cell differentiation, and the acute phase response. It is essential for studying mechanisms underlying autoimmune diseases, infection responses, and tissue regeneration.

• Disease Pathogenesis Studies: Elevated IL-6 levels are implicated in a variety of disease states, including diabetes, rheumatoid arthritis, cancer, and neuroinflammatory conditions. Recombinant rat IL-6 enables controlled investigation of its role in disease progression and therapeutic intervention.

• Cellular and Molecular Mechanism Research: IL-6 signals through the IL-6 receptor and GP130, activating downstream pathways such as STAT3, which are crucial for cell proliferation, survival, and differentiation. Recombinant protein allows precise manipulation of these pathways in vitro and in vivo.

• Hybridoma and Cell Line Growth: IL-6 is commonly used to support the growth of hybridoma cell lines and other immune cells in culture, facilitating antibody production and immunological assays.

• High Purity and Bioactivity: Recombinant rat IL-6 is typically expressed in E. coli and purified to high standards, ensuring reproducible and reliable results in experimental systems. It has been shown to be highly bioactive, sometimes exceeding the activity of human IL-6 in rat-specific assays.

• Preclinical Therapeutic Studies: Recombinant rat IL-6 is valuable for testing anti-cytokine therapies, such as receptor fusion proteins or monoclonal antibodies, in rodent models of inflammation and autoimmunity.

• Neuroscience and Stress Research: IL-6 is involved in CNS inflammation and can modulate synaptic activity, making it relevant for studies on neuroinflammation, stress, and neurodegenerative diseases.

In summary, recombinant rat IL-6 is an essential tool for dissecting cytokine biology, modeling disease mechanisms, and developing therapeutic strategies in rat-based research systems. Its use ensures experimental consistency, species specificity, and high biological activity for a wide range of immunological and biomedical applications.

Yes, recombinant rat IL-6 is specifically designed and validated for use as a standard for quantification and calibration in ELISA assays. This is one of the primary applications of recombinant rat IL-6 proteins in research.

Validation and Accuracy

The recombinant rat IL-6 produced through E. coli expression systems has been extensively validated for ELISA standardization. Studies have demonstrated that dose-response curves obtained using natural rat IL-6 are parallel to standard curves generated with recombinant rat IL-6 standards, confirming the accuracy and reliability of recombinant protein for calibration purposes. This parallelism indicates that the recombinant standard accurately reflects the behavior of endogenous IL-6 in biological samples.

Typical Calibration Range

ELISA kits utilizing recombinant rat IL-6 standards typically employ calibration ranges spanning from approximately 10-4000 pg/mL, depending on the specific assay platform. The undiluted recombinant standard typically serves as the high calibration point, while diluted standards create intermediate calibration points for constructing the standard curve.

Recommended Applications

Recombinant rat IL-6 is particularly suitable for use as an ELISA standard in the following contexts:

• Cell culture supernatant analysis - quantifying IL-6 secretion from cultured cells
• Serum and plasma measurements - determining circulating IL-6 levels in biological samples
• Tissue homogenate analysis - measuring IL-6 in tissue preparations

Formulation Considerations

When selecting recombinant rat IL-6 for use as an ELISA standard, formulations containing bovine serum albumin (BSA) are generally recommended for optimal performance in assay applications. The BSA helps stabilize the protein and prevents non-specific binding to assay surfaces.

Recombinant Rat IL-6 has been validated for several key applications in published research, primarily in immunological, cell biology, and disease modeling studies.

Validated Applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or analyte to quantify IL-6 levels in biological samples.
• Western Blot: Applied to detect IL-6 protein expression and confirm antibody specificity.
• Functional Assays: Extensively used to study cellular responses, such as proliferation, differentiation, and cytokine signaling in various cell types including hepatocytes, B and T lymphocytes, neurons, tumor cells, and hematopoietic cells.
• Immunoprecipitation: Utilized to isolate IL-6 or its interacting partners from cell lysates.
• Immunohistochemistry: Employed to localize IL-6 protein in tissue sections.
• Cell Culture Supplementation: Used to stimulate or modulate immune cell function, support hybridoma growth, and investigate inflammatory pathways.
• Disease Modeling: Applied in studies of inflammatory diseases such as rheumatoid arthritis, Kaposi sarcoma, systemic juvenile arthritis, and peripheral nerve injury.

Supporting Details:

• Recombinant rat IL-6 is a multifunctional cytokine involved in immune response, inflammation, and hematopoiesis.
• It has been used to decipher cellular responses, develop potential therapeutics, and study cytokine signaling mechanisms.
• In vivo studies include direct injection into tissues (e.g., sciatic nerve) to assess local effects on inflammation and tissue response.
• It is commonly used as a positive control or standard in immunoassays and as a stimulant in cell-based assays to investigate downstream signaling and gene expression.

Additional Notes:

• Recombinant rat IL-6 is not approved for clinical diagnostic use; it is strictly for research applications.
• Its activity has been confirmed across a range of cell types and experimental systems, supporting its broad utility in immunological and biomedical research.

In summary, recombinant rat IL-6 is validated for ELISA, Western blot, functional assays, immunoprecipitation, immunohistochemistry, cell culture supplementation, and disease modeling, with extensive use in studies of immune regulation and inflammation.

To reconstitute and prepare Recombinant Rat IL-6 protein for cell culture experiments, dissolve the lyophilized protein in an acidic buffer (such as 10 mM HCl or 100 mM acetic acid) or sterile water/buffer at neutral to slightly basic pH, then dilute into cell culture medium or assay buffer as needed. The specific protocol may vary depending on the protein’s formulation and intended use.

General Protocol for Reconstitution and Preparation:

• Warming: Allow the lyophilized vial to equilibrate to room temperature before opening to minimize condensation.
• Reconstitution Buffer:
  • If the product is lyophilized from an acidic solution (e.g., 0.1% TFA), reconstitute in 10 mM HCl or 100 mM acetic acid to a concentration of 0.1–1 mg/mL.
  • Alternatively, some protocols recommend reconstitution in sterile distilled water or buffer at pH >7 (e.g., PBS or Tris buffer) to a concentration of 100 ng/μL (0.1 mg/mL).
• Mixing: Gently pipette up and down or swirl to dissolve. Avoid vigorous vortexing to prevent protein denaturation.
• Incubation: Let the solution sit at room temperature for 5–30 minutes to ensure complete solubilization.
• Aliquoting: Divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Store aliquots at -20°C to -80°C for long-term stability.
  • Short-term storage (up to 1 week) can be at 2–8°C.
• Dilution for Cell Culture:
  • Before adding to cells, dilute the stock solution into cell culture medium or assay buffer to the desired working concentration.
  • If the protein was reconstituted in acid, neutralize by further dilution into medium or buffer containing serum or BSA to prevent cell toxicity.

Key Considerations:

• Use sterile, low-protein-binding tubes for all steps to minimize adsorption and contamination.
• If the protein is sensitive to oxidation or aggregation, consider adding carrier protein (e.g., 0.1% BSA) to the dilution buffer.
• Avoid repeated freeze-thaw cycles by aliquoting immediately after reconstitution.

Example Protocol:

1. Add 100 μL of 10 mM HCl to 10 μg lyophilized IL-6 to achieve 0.1 mg/mL.
2. Gently mix and incubate at room temperature for 15–30 minutes.
3. Aliquot and store at -80°C.
4. For cell culture, dilute the stock into pre-warmed culture medium to the desired final concentration (e.g., 1–100 ng/mL), ensuring the final pH is neutral.

Summary Table:

Always consult the specific product datasheet for any manufacturer-specific recommendations, as formulations may differ. If using for cell-based assays, ensure the final solution is compatible with your cells and does not contain residual acid at cytotoxic levels.