Recombinant Mouse TNF-α

Recombinant Mouse TNFα is widely used in research due to its central role as a proinflammatory 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 TNFα in research applications include:

• Controlled and reproducible experimental conditions: Recombinant TNFα 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: TNFα is used to stimulate antigenpresenting 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 TNFα is essential for probing TNFα–induced intracellular signaling cascades, such as NFκB 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 TNFα 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 antiTNF therapies in autoimmune and inflammatory diseases, and is used to evaluate the efficacy and mechanism of candidate therapeutics.

• Speciesspecific relevance: Using mouse TNFα is crucial for murine models, ensuring physiological relevance and compatibility with mouse immune system components.

In summary, recombinant Mouse TNFα is a versatile and indispensable tool for dissecting immune mechanisms, modeling disease, and developing therapeutics in mousebased research systems.

Yes, recombinant mouse TNFα is widely used as a standard for quantification or calibration in ELISA assays. This practice is wellestablished and supported by multiple technical sources and protocols.

Key points and best practices:

• Recombinant mouse TNFα is specifically recommended as an ELISA standard for quantifying mouse TNFα in biological samples. This is standard in both commercial kits and custom assay development.
• Assay compatibility: Most mouse TNFα ELISA kits are validated to detect both recombinant and natural forms of TNFα, with parallel doseresponse 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 TNFα in unknown samples.
• Formulation considerations: For ELISA, recombinant TNFα is often supplied with or without carrier proteins (e.g., BSA). Carrierfree 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 TNFα, 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 TNFα as ELISA Standard

Additional recommendations:

• Confirm the source and sequence of your recombinant TNFα 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 inhouse ELISA, validate the assay’s linearity, sensitivity, and specificity with the recombinant standard before quantifying unknowns.

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

Recombinant Mouse TNFα has been validated for a broad range of applications in published research, primarily in cellbased assays, immunoassays, and functional studies involving immune modulation and cytotoxicity.

Key validated applications include:

• Bioassays for Cytotoxicity and Cell Proliferation:
  Recombinant mouse TNFα is widely used to induce cytolysis in sensitive cell lines such as L929 and WEHI13VAR, serving as a standard for measuring TNFα activity and for screening TNFα inhibitors.

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

• Assessment of Apoptosis and Cell Death Pathways:
  TNFα 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 TNFα–Induced Signaling Pathways:
  Researchers use recombinant mouse TNFα to dissect downstream signaling events, such as NFκB activation, MAPK pathways, and inflammatory gene expression.

• ELISA and Immunoassay Standards:
  Recombinant mouse TNFα serves as a standard in ELISA for quantifying TNFα 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., LPSinduced sepsis, TPAinduced edema), to study disease mechanisms, and to validate antiTNFα therapeutics.

• Cell Culture Supplementation:
  TNFα 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 TNFα stimulation.

• Therapeutic Antibody Validation:
  Used as a target in the development and validation of TNFα 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 peerreviewed studies, confirming the versatility and reliability of recombinant mouse TNFα in immunology, cell biology, and translational research.

Reconstitution Protocol

Recombinant mouse TNFα 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 longterm 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 freezethaw cycles, which can significantly reduce protein activity and cause aggregation
• If multiple aliquots are needed, prepare them during initial reconstitution to minimize freezethaw 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.