Recombinant Mouse TNFα (aa 84-235)

Recombinant Mouse TNFα (aa 84-235) is widely used in research because it represents the biologically active extracellular domain of TNFα, enabling precise investigation of TNFα-mediated signaling, inflammation, and immune responses in mouse models and cell-based assays.

Key scientific reasons to use this construct:

• Biologically Active Domain: The aa 84-235 fragment corresponds to the mature, soluble extracellular portion of mouse TNFα, which is responsible for binding to TNF receptors (TNFR1 and TNFR2) and initiating downstream signaling pathways relevant to inflammation, apoptosis, and immune modulation.
• High Bioactivity: The truncated aa 84-235 form has been shown to possess a higher degree of bioactivity in bioassays compared to slightly longer constructs (e.g., aa 80-235), making it particularly suitable for functional studies requiring robust TNFα activity.
• Standardization: Recombinant TNFα (aa 84-235) provides a consistent, well-characterized reagent for reproducible results in cell culture, differentiation studies, ELISA standards, and functional assays.
• Versatile Applications: It is used to:
  • Induce osteoclastogenesis and study bone metabolism.
  • Activate antigen-presenting cells and assess immune cell function.
  • Investigate cytotoxicity, apoptosis, and anti-tumor responses.
  • Model inflammatory processes and cytokine signaling in vitro and in vivo.
• Species Specificity: Mouse TNFα is essential for experiments involving mouse cells or mouse models, ensuring physiological relevance and compatibility with mouse TNF receptors.

Experimental considerations:

• Formulation: Carrier-free or BSA-containing formulations are available; choose based on downstream application (e.g., avoid BSA for mass spectrometry or applications sensitive to carrier proteins).
• Purity and Endotoxin: Recombinant preparations are typically highly purified and tested for low endotoxin levels, minimizing confounding effects in sensitive assays.

Summary of scientific applications:

• Cell signaling studies (NF-κB, apoptosis, cytokine networks)
• Immunology research (macrophage activation, T cell responses)
• Bone biology (osteoclast differentiation, bone resorption)
• Cancer biology (tumor cell cytotoxicity, immune modulation)
• Inflammation models (in vitro and in vivo)

Using Recombinant Mouse TNFα (aa 84-235) ensures you are working with the active, physiologically relevant form of TNFα for mouse-based research, supporting robust and reproducible experimental outcomes.

You can use recombinant mouse TNFα (aa 84-235) as a standard for quantification or calibration in ELISA assays, provided that the ELISA kit you are using is validated to recognize this specific fragment and that the standard curve is prepared in the same matrix and diluent as recommended by the kit protocol.

Key considerations and supporting details:

• ELISA kits are typically calibrated using recombinant mouse TNFα, often produced in E. coli and covering the mature protein sequence, which generally spans amino acids 80–235 or similar. Your protein (aa 84–235) closely matches this region, differing only by a few N-terminal residues, which are not generally critical for antibody recognition in most commercial sandwich ELISAs.

• Commercial ELISA kits and protocols specify that both recombinant and natural mouse TNFα are recognized equivalently, and dose-response curves for both forms are parallel, indicating that recombinant standards are suitable for quantification.

• Critical factors for use as a standard:

  • The antibodies in your ELISA must recognize epitopes present within aa 84–235. Most commercial kits use antibodies raised against the mature region, which your protein covers.
  • Standard preparation: Follow the kit’s instructions for reconstitution and dilution, using the recommended diluent to ensure matrix compatibility and accurate quantification.
  • Bioactivity and purity: The recombinant TNFα (aa 84–235) is reported to have high bioactivity in bioassays. Ensure your preparation is pure and correctly quantified for reliable standard curve generation.

• Potential caveats:

  • If your ELISA kit is calibrated against a different TNFα isoform (e.g., aa 80–235 or full-length), there may be minor differences in absolute quantification, but these are typically negligible for most applications.
  • If you are developing a custom ELISA, validate that your capture and detection antibodies bind the aa 84–235 region without loss of sensitivity or specificity.

Summary:
Recombinant mouse TNFα (aa 84–235) is suitable as a standard for ELISA quantification if it matches the epitope region recognized by your assay’s antibodies and is prepared according to the kit’s protocol. This approach is widely accepted and used in both commercial and custom ELISA systems. Always verify compatibility with your specific assay to ensure accurate results.

Recombinant Mouse TNFα (aa 84-235) has been validated primarily for in vitro bioactivity assays in published research, especially in cell culture systems to study TNFα-mediated signaling, inflammation, apoptosis, and osteoclastogenesis.

Key validated applications in published research include:

• Bioactivity assays: Used to stimulate mouse cells in vitro to assess downstream signaling, cytokine production, apoptosis, and other TNFα-dependent cellular responses.
• Cell culture experiments: Applied to cultured cells to model inflammatory responses, immune cell activation, and osteoclast differentiation.
• Osteoclastogenesis studies: Specifically used in bioassays to induce osteoclast differentiation via TNFα signaling, as demonstrated in multiple peer-reviewed studies.
• Functional studies of TNFα signaling: Used to dissect the role of TNFα in various cellular pathways, including apoptosis, immune modulation, and metabolic regulation.

Supporting details:

• The protein is a truncated, soluble form corresponding to the extracellular domain, optimized for high bioactivity in vitro.
• Published studies have used this reagent to investigate TNFα-induced effects in mouse whole cell systems, including podocyte ER stress, osteoclastogenesis, and receptor signaling.
• Vendor and technical datasheets, as well as peer-reviewed literature, consistently cite bioactivity validation as the primary application.

Not validated for:

• In vivo studies (such as direct animal injection) or as a standard in immunoassays (e.g., ELISA) are not specifically cited for the aa 84-235 fragment; these applications are more commonly validated for the slightly longer aa 80-235 variant.

Summary Table:

For any application beyond in vitro bioactivity (such as in vivo use or as an ELISA standard), validation should be confirmed for the specific fragment and context.

To reconstitute and prepare Recombinant Mouse TNFα (aa 84-235) for cell culture experiments, dissolve the lyophilized protein at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). This helps stabilize the protein and prevent adsorption to surfaces.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS with at least 0.1% BSA to the vial to achieve a final concentration of 50 μg/mL.
  • Gently mix by pipetting or swirling; avoid vigorous agitation to prevent denaturation.
  • If the protein appears as a film, ensure complete dissolution by gentle mixing.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can reduce activity.
  • Store aliquots at –20°C to –70°C for long-term storage, or at 2–8°C for up to one month under sterile conditions.
  • For extended storage, always include carrier protein (BSA) in the buffer to maintain stability.

• Working Solution Preparation:

  • For cell culture, further dilute the stock solution in your culture medium immediately before use.
  • Ensure the final working concentration is optimized for your specific assay, as TNFα activity is dose-dependent.

Additional notes:

• Do not reconstitute at concentrations above 1 mg/mL to avoid solubility issues.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Use sterile technique throughout to prevent contamination.

This protocol ensures maximum stability and bioactivity of recombinant mouse TNFα (aa 84-235) for cell culture applications.