Recombinant Mouse TNF-α

Recombinant Mouse TNF-α is widely used in research due to its central role as a pro-inflammatory cytokine that regulates immune responses, cell death, and tissue homeostasis, making it essential for studies in immunology, inflammation, cancer, and drug development.

Key scientific reasons to use recombinant Mouse TNF-α in research applications include:

• Controlled and reproducible experimental conditions: Recombinant TNF-α provides a consistent, defined source of cytokine, eliminating variability associated with endogenous or crude preparations. This is critical for quantitative assays and mechanistic studies.

• Activation and modulation of immune cells: TNF-α is used to stimulate antigen-presenting cells, induce differentiation, and activate neutrophils, monocytes, and T cells, allowing researchers to dissect immune cell functions and signaling pathways.

• Functional assays: It is employed in cytotoxicity, cell proliferation, apoptosis, and viral protection assays to study cell fate decisions and immune defense mechanisms.

• Investigation of signaling pathways: Recombinant TNF-α is essential for probing TNF-α–induced intracellular signaling cascades, such as NF-κB and MAPK, which regulate inflammation, apoptosis, and immune regulation.

• Disease modeling: It is used to model inflammatory diseases (e.g., arthritis, diabetes), cancer progression, and tissue injury in vitro and in vivo, enabling the study of pathogenesis and therapeutic interventions.

• Standardization in analytical assays: Recombinant TNF-α serves as a positive control or standard in ELISA, western blotting, and bioassays, ensuring assay reliability and comparability.

• Therapeutic research: It is a target for drug development, especially for anti-TNF therapies in autoimmune and inflammatory diseases, and is used to evaluate the efficacy and mechanism of candidate therapeutics.

• Species-specific relevance: Using mouse TNF-α is crucial for murine models, ensuring physiological relevance and compatibility with mouse immune system components.

In summary, recombinant Mouse TNF-α is a versatile and indispensable tool for dissecting immune mechanisms, modeling disease, and developing therapeutics in mouse-based research systems.

Yes, recombinant mouse TNF-α is widely used as a standard for quantification or calibration in ELISA assays. This practice is well-established and supported by multiple technical sources and protocols.

Key points and best practices:

• Recombinant mouse TNF-α is specifically recommended as an ELISA standard for quantifying mouse TNF-α in biological samples. This is standard in both commercial kits and custom assay development.
• Assay compatibility: Most mouse TNF-α ELISA kits are validated to detect both recombinant and natural forms of TNF-α, with parallel dose-response curves, ensuring accurate quantification.
• Calibration and quantification: The recombinant standard is serially diluted to generate a standard curve, which is then used to interpolate the concentration of TNF-α in unknown samples.
• Formulation considerations: For ELISA, recombinant TNF-α is often supplied with or without carrier proteins (e.g., BSA). Carrier-free formulations are preferred if BSA or other additives could interfere with your assay.
• Standard preparation: Always prepare fresh dilutions of the recombinant standard for each assay, as stability can be limited after reconstitution.
• Validation: Ensure that your ELISA antibodies recognize both recombinant and native TNF-α, and that the standard curve generated with the recombinant protein is parallel to the response for your sample matrix.

Summary Table: Use of Recombinant Mouse TNF-α as ELISA Standard

Additional recommendations:

• Confirm the source and sequence of your recombinant TNF-α matches the native protein recognized by your assay antibodies.
• Follow the manufacturer’s instructions for reconstitution, dilution, and storage of the recombinant standard.
• If using a custom or in-house ELISA, validate the assay’s linearity, sensitivity, and specificity with the recombinant standard before quantifying unknowns.

In summary: Recombinant mouse TNF-α is appropriate and commonly used as a standard for ELISA quantification and calibration, provided the assay is validated for this application.

Recombinant Mouse TNF-α has been validated for a broad range of applications in published research, primarily in cell-based assays, immunoassays, and functional studies involving immune modulation and cytotoxicity.

Key validated applications include:

• Bioassays for Cytotoxicity and Cell Proliferation:
  Recombinant mouse TNF-α is widely used to induce cytolysis in sensitive cell lines such as L929 and WEHI-13VAR, serving as a standard for measuring TNF-α activity and for screening TNF-α inhibitors.

• Activation of Antigen Presenting Cells:
  It is used to stimulate dendritic cells, macrophages, and other antigen-presenting cells to study immune activation and cytokine signaling.

• Assessment of Apoptosis and Cell Death Pathways:
  TNF-α is employed to trigger apoptosis, necroptosis, and pyroptosis in various cell types, enabling the study of cell death mechanisms and related signaling pathways.

• Investigation of TNF-α–Induced Signaling Pathways:
  Researchers use recombinant mouse TNF-α to dissect downstream signaling events, such as NF-κB activation, MAPK pathways, and inflammatory gene expression.

• ELISA and Immunoassay Standards:
  Recombinant mouse TNF-α serves as a standard in ELISA for quantifying TNF-α levels in biological samples, and as a positive control in western blotting.

• In Vivo and Ex Vivo Functional Studies:
  It is used in mouse models to induce inflammatory responses (e.g., LPS-induced sepsis, TPA-induced edema), to study disease mechanisms, and to validate anti-TNF-α therapeutics.

• Cell Culture Supplementation:
  TNF-α is added to cell cultures to modulate immune responses, study cytokine interactions, and assess the effects of genetic or pharmacological interventions.

Representative published research applications:

• Bioassays in Disease Models:
  Used to study lung cancer sex bias, neurodegeneration, autoimmune disorders, and metabolic disease in mouse models by modulating inflammatory responses.

• Mechanistic Studies:
  Applied to investigate mitochondrial function, mitophagy, and immune cell migration in response to TNF-α stimulation.

• Therapeutic Antibody Validation:
  Used as a target in the development and validation of TNF-α inhibitors, including nanobodies and monoclonal antibodies, in both in vitro and in vivo systems.

Summary Table of Validated Applications

These applications are supported by both product validation data and numerous peer-reviewed studies, confirming the versatility and reliability of recombinant mouse TNF-α in immunology, cell biology, and translational research.

Reconstitution Protocol

Recombinant mouse TNF-α is typically supplied as lyophilized material and requires proper reconstitution before use in cell culture experiments. The standard reconstitution concentration is 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). Some formulations may specify alternative reconstitution buffers, such as sterile water, depending on the specific product formulation.

Key reconstitution steps:

• Allow the lyophilized vial to warm to room temperature before opening to minimize condensation
• Add the appropriate volume of reconstitution buffer to achieve the desired concentration
• Mix gently but thoroughly; the protein may initially appear as a film at the bottom of the vial, so careful mixing is essential
• Allow the solution to sit for approximately 5 minutes at room temperature before use

Storage and Stability Considerations

Proper storage is critical for maintaining protein activity and preventing degradation. Store reconstituted protein at -20°C or -80°C (with -80°C being the recommended long-term storage temperature). The lyophilized powder itself is highly stable at -20°C.

Important storage guidelines:

• Use a manual defrost freezer to avoid temperature fluctuations
• Avoid repeated freeze-thaw cycles, which can significantly reduce protein activity and cause aggregation
• If multiple aliquots are needed, prepare them during initial reconstitution to minimize freeze-thaw exposure
• Store aliquots in polypropylene vials rather than glass when possible

Sample Preparation for Cell Culture

When preparing samples for cell culture experiments, handle them with care to maintain protein integrity. Remove any particulate matter by centrifugation before use. If samples require dilution, prepare serial dilutions in appropriate culture medium or assay diluent immediately before the experiment.

For experiments involving high cytokine concentrations, prepare multiple dilutions to ensure measurements fall within the standard curve range. This is particularly important for accurate quantification in downstream assays such as ELISA or bioassays.