Using Recombinant Mouse IL-12 (HEK293 Expressed) in research offers several key advantages, particularly for immunological studies, cell signaling assays, and preclinical therapeutic development. The choice of HEK293 as the expression system ensures mammalian post-translational modifications, high protein quality, and reduced risk of non-murine glycosylation artifacts.

Key reasons to use Recombinant Mouse IL-12 (HEK293 Expressed):

• Mammalian Post-Translational Modifications (PTMs): HEK293 cells, being human-derived, provide complex PTMs such as glycosylation and proper protein folding, which are critical for the biological activity and stability of cytokines like IL-12. This is especially important for functional studies, as non-mammalian systems (e.g., E. coli) may produce proteins lacking these modifications, potentially altering activity or immunogenicity.

• High Biological Activity: Recombinant mouse IL-12 produced in HEK293 cells is biologically active and can effectively stimulate immune responses, such as the differentiation of Th1 cells and induction of IFN-γ production, which are central to studies of cellular immunity, tumor immunology, and infectious disease models.

• Species Compatibility: Mouse IL-12 is active on both mouse and human cells, making it suitable for cross-species experiments, including murine models and humanized systems.

• Reduced Immunogenicity: Proteins expressed in HEK293 cells are less likely to contain non-human glycan epitopes (such as Neu5Gc or α-Gal), which can reduce immunogenicity and improve translational relevance for in vivo studies.

• Rapid and Scalable Production: HEK293 cells are highly transfectable and support both transient and stable expression, allowing for quick production of recombinant proteins at laboratory and preclinical scales.

• Consistency and Reproducibility: HEK293-expressed proteins offer batch-to-batch consistency, which is critical for reproducible experimental results.

Typical research applications include:

• Immunological assays: Studying T cell differentiation, NK cell activation, and cytokine signaling pathways.
• Cancer immunotherapy research: Evaluating IL-12’s effects on tumor models, immune cell infiltration, and combination therapies.
• Cell signaling studies: Dissecting STAT4 activation and downstream gene expression in response to IL-12 stimulation.
• Preclinical therapeutic development: Testing IL-12-based biologics or immunocytokines in murine models.

Summary Table: HEK293-Expressed Mouse IL-12 vs. Other Systems

In summary, Recombinant Mouse IL-12 (HEK293 Expressed) is preferred for applications requiring high biological activity, native protein structure, and translational relevance, especially in immunology and preclinical research.

You can use recombinant mouse IL-12 expressed in HEK293 cells as a standard for quantification or calibration in your ELISA assays, provided it matches the form of IL-12 your assay is designed to detect (e.g., p70 heterodimer or p40 subunit) and is validated for this use by the assay protocol or manufacturer.

Key considerations and supporting details:

• Form of IL-12: Mouse IL-12 exists as a heterodimer (p70, composed of p35 and p40 subunits) or as the p40 subunit alone. Ensure your recombinant standard matches the analyte your ELISA detects (most ELISAs for IL-12 are specific for the p70 heterodimer, but some detect p40).
• Expression System: Recombinant IL-12 produced in HEK293 cells is commonly used as a standard in ELISA kits and is considered suitable for quantification, as it closely mimics the natural protein’s post-translational modifications.
• Validation: Commercial ELISA kits for mouse IL-12 (p70) routinely use recombinant mouse IL-12 (often HEK293-expressed) as the standard and demonstrate parallelism between recombinant and natural IL-12 in standard curves.
• Standard Curve Preparation: Prepare a fresh standard curve with each assay run, using serial dilutions of the recombinant protein in the same buffer/matrix as your samples for accurate quantification.
• Activity and Purity: Confirm that the recombinant protein is bioactive and of high purity (typically >95%) for use as a standard.
• Documentation: Check the product datasheet or technical documentation for explicit mention of suitability as an ELISA standard. Most reputable suppliers provide this information.

Best Practices:

• Use the same diluent for standards and samples to minimize matrix effects.
• Validate the standard curve for linearity and parallelism with your sample matrix if using a custom or non-kit standard.
• If your ELISA is designed for the p70 heterodimer, do not use p40-only or p35-only standards, as these will not accurately reflect the analyte detected by the assay.

In summary, recombinant mouse IL-12 (HEK293-expressed) is widely accepted and validated as a standard for ELISA quantification, provided it matches the analyte detected by your assay and is of high purity and activity. Always consult your specific ELISA protocol for compatibility and validation requirements.

Applications of Recombinant Mouse IL-12 (HEK293 Expressed)

Recombinant mouse IL-12 expressed in HEK293 cells has been validated for several key applications in research and development:

Immunological Assays

Functional bioactivity assays represent a primary application for recombinant mouse IL-12. The biological activity of these proteins is typically determined through measurement of induced immune responses, such as CD25 expression on CD8+ mouse T cells or interferon-gamma (IFN-γ) production by IL-12-responsive cell lines. This validation confirms that the recombinant protein maintains its capacity to stimulate appropriate immune cell populations.

Analytical and Biochemical Applications

The protein has been validated for use in SDS-PAGE and sandwich ELISA (sELISA) applications. These techniques enable researchers to characterize protein purity, molecular weight, and quantify IL-12 levels in experimental samples. High purity specifications (typically >95% by SDS-PAGE and HPLC) support reliable results in these analytical methods.

Therapeutic and Drug Development

Recombinant mouse IL-12 serves important roles in therapeutic development and drug screening applications. The protein's validated bioactivity makes it suitable for preclinical studies investigating IL-12-based immunotherapeutics and for screening compounds that modulate IL-12 signaling pathways.

Immunological Research Context

The functional validation of these proteins is grounded in IL-12's well-characterized biological roles: promoting Th1 cell differentiation, stimulating T cell growth and function, enhancing IFN-γ and TNF-α production, and contributing to antimycobacterial immune responses. These validated applications leverage the protein's ability to engage IL-12 receptor signaling and trigger downstream immune activation.

To reconstitute and prepare Recombinant Mouse IL-12 (HEK293 Expressed) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a recommended concentration, then dilute as needed for your assay.

Essential protocol details:

• Reconstitution:
  Add sterile, distilled water to the lyophilized IL-12 protein to achieve a concentration of 0.1 mg/mL (100 μg/mL). If your product datasheet specifies a different buffer (e.g., PBS, pH 7.2–7.5), use that instead. Gently mix by pipetting; avoid vigorous vortexing to prevent protein denaturation.

• Sterility:
  Ensure all solutions and containers are sterile. Use a biosafety cabinet if possible.

• Dilution for cell culture:
  After reconstitution, further dilute the stock solution in cell culture medium or assay buffer to the desired working concentration. Typical final concentrations for functional assays range from 0.04–1 ng/mL, depending on cell type and experimental design.

• Storage:
  Aliquot the reconstituted stock to avoid repeated freeze-thaw cycles. Store at –20°C or –80°C for long-term use. Avoid storing at 4°C for extended periods, as this may reduce activity.

• Endotoxin:
  Confirm the endotoxin level is suitable for cell culture (<0.005 EU/μg is typical for high-quality recombinant cytokines).

Best practices for cell culture experiments:

• Use serum-free or low-serum medium if possible to minimize background cytokine activity.
• Include appropriate controls (e.g., media only, positive control cytokine).
• For stimulation assays, add IL-12 to cells and incubate for 16–24 hours before measuring downstream effects (e.g., IFN-γ secretion).
• Avoid repeated freeze-thaw cycles of the protein stock to maintain bioactivity.

Summary Table: Preparation Steps

Always consult the specific product datasheet for any manufacturer-specific recommendations regarding buffer composition, reconstitution volume, and storage conditions.