Recombinant Mouse sTNF RI

Recombinant Mouse sTNF RI is used in research applications primarily to neutralize the biological activity of mouse TNF-α and TNF-β, thereby modulating inflammatory responses in vitro and in vivo. This protein acts as a decoy receptor, binding soluble TNF and preventing its interaction with cell surface TNF receptors, which are key mediators of inflammation and cell death.

Key scientific applications and advantages include:

• Anti-inflammatory studies: sTNF RI is widely used to investigate the role of TNF signaling in inflammatory diseases, such as rheumatoid arthritis, heart failure, and viral infections. By blocking TNF-α, it allows researchers to dissect the contribution of TNF to disease pathology and evaluate therapeutic strategies.
• Mechanistic studies: sTNF RI helps elucidate the downstream effects of TNF inhibition, such as suppression of NFκB activation, reduction of pro-inflammatory cytokine production, and improvement of tissue pathology in models of cardiac and pulmonary inflammation.
• Modeling therapeutic interventions: Recombinant sTNF RI mimics the action of clinically relevant anti-TNF biologics, enabling preclinical testing of anti-TNF strategies in mouse models before translation to human studies.
• Species specificity: Using mouse sTNF RI ensures optimal activity and compatibility in murine systems, as TNF and its receptors exhibit species-specific interactions. Mouse cells respond more effectively to homologous (mouse-derived) TNF inhibitors than to human versions.
• Functional assays: sTNF RI is used in cell-based assays to neutralize TNF-mediated cytotoxicity, such as in L-929 cell assays, providing a quantitative measure of TNF activity and inhibition.

Additional relevant details:

• Developmental biology: TNF RI is essential for proper lymphoid organ development and immune regulation in mice.
• Paracrine signaling: sTNF RI is a major mediator of mesenchymal stem cell (MSC) anti-inflammatory effects, indicating its importance in cell therapy research.
• Versatility: Recombinant mouse sTNF RI can be used in a variety of experimental setups, including acute and chronic inflammation models, infectious disease studies, and immune regulation research.

In summary, recombinant mouse sTNF RI is a critical tool for dissecting TNF-dependent pathways, modeling anti-TNF therapies, and controlling inflammation in mouse-based research applications. Its use is essential for studies requiring species-specific reagents and for accurately modeling therapeutic interventions targeting TNF signaling.

Yes, recombinant mouse sTNF RI can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and formulated appropriately for this application.

Key considerations and supporting details:

• Intended Use: Recombinant mouse sTNF RI (soluble TNF receptor I, TNFRSF1A) is commonly used as a standard in ELISA assays to generate a standard curve for quantifying sTNF RI in biological samples. Many ELISA kits and protocols specifically recommend or supply recombinant protein standards for this purpose.

• Formulation: For use as an ELISA standard, it is generally advised to use the recombinant protein formulated with a carrier protein such as BSA, which helps stabilize the protein and prevent adsorption to plastic surfaces. Carrier-free formulations are typically reserved for applications where BSA or other carriers may interfere with downstream assays.

• Purity and Activity: The recombinant standard should be highly purified and its concentration accurately determined. It should also be validated for immunoreactivity with the antibodies used in your ELISA to ensure parallel recognition of both the recombinant standard and the native protein in samples.

• Parallelism: Well-designed ELISA kits and protocols confirm that the antibody pairs recognize both the recombinant standard and the natural protein in biological samples in a parallel manner, ensuring accurate quantification. This is critical for reliable calibration.

• Documentation: Always consult the technical datasheet or product documentation for your specific recombinant sTNF RI preparation to confirm its suitability as an ELISA standard. Some recombinant proteins are specifically tested and validated for use as ELISA standards, while others may not be.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant sTNF RI in the same buffer or matrix as your samples to generate a standard curve for quantification. Follow best practices to avoid technical errors in standard preparation.

Summary Table: Use of Recombinant Mouse sTNF RI as ELISA Standard

In summary: If your recombinant mouse sTNF RI is validated for ELISA and formulated appropriately, it is suitable for use as a standard in quantification or calibration of ELISA assays. Always verify with the product datasheet and, if possible, run parallelism tests to confirm equivalence with the native analyte in your samples.

Recombinant Mouse sTNF RI (soluble TNF Receptor I, TNFRSF1A) has been validated for several key applications in published research, most notably as an ELISA standard, in bioassays, and for cytokine inhibition studies.

Validated Applications in Published Research:

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Used as a standard for quantifying soluble TNF RI in biological samples, enabling measurement of cytokine/inhibitor profiles in mouse models of sepsis and inflammation.
  • Sequential ELISA protocols have utilized recombinant sTNF RI to profile multiple cytokines from small sample volumes.

• Bioassay:

  • Applied in functional assays to assess TNF-α activity and inhibition, including studies of immune modulation and inflammatory responses.

• Cytokine Inhibition Studies:

  • Used to directly inhibit TNF-α signaling in mouse models, allowing investigation of downstream effects such as changes in VEGF-C and VEGFR-3 expression, and the impact on disease severity in models of acquired lymphedema.
  • Demonstrated utility in studies examining the role of TNF-α in tissue repair and immune regulation.

• Cell and Tissue Culture:

  • Recommended for use in cell culture experiments to study TNF-α signaling, apoptosis, and immune cell responses.

• Western Blot:

  • Cited as suitable for use in Western blot protocols to detect TNF RI protein levels in cell lysates.

Research Areas and Experimental Contexts:

• Immunology and Inflammation: Frequently used to study TNF-α-mediated immune responses, apoptosis, and inflammatory signaling pathways.
• Innate Immunity: Applied in experiments investigating the role of TNF RI in innate immune cell function and cytokine signaling.
• Disease Models: Utilized in mouse models of sepsis, lymphedema, and autoimmune disease to dissect the role of TNF-α and its receptors in pathogenesis.

Summary Table of Validated Applications

Key Notes:

• Recombinant Mouse sTNF RI is widely used as a tool to dissect TNF-α signaling and its biological consequences in mouse models.
• Its role as a soluble inhibitor of TNF-α makes it valuable for both mechanistic studies and as a control reagent in cytokine quantification assays.
• Published research supports its use in both in vitro and in vivo experimental setups, particularly in immunology and inflammation research.

If you require protocol details or specific experimental setups, please specify the application of interest.

To reconstitute and prepare Recombinant Mouse sTNF RI (soluble TNF Receptor I, TNFRSF1A) protein for cell culture experiments, follow these best-practice steps:

1. Reconstitution Buffer Selection

• Use sterile PBS (phosphate-buffered saline) as the primary reconstitution buffer.
• If the protein is carrier-free, consider adding a carrier protein such as 0.1% BSA (bovine serum albumin) to minimize adsorption and stabilize the protein, especially for low-concentration or long-term storage.

2. Reconstitution Procedure

• Allow the lyophilized protein vial to equilibrate to room temperature before opening to prevent condensation.
• Add the recommended volume of sterile PBS (with or without carrier protein) to achieve the desired stock concentration. Common concentrations are 100–200 μg/mL for stock solutions.
• Gently swirl or invert the vial to dissolve. Avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
• Let the solution sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

3. Aliquoting and Storage

• Once fully dissolved, aliquot the stock solution into small volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at –20°C to –70°C for long-term storage, or at 2–8°C for short-term use (up to 1 month).
• Avoid multiple freeze-thaw cycles by using single-use aliquots.

4. Preparation for Cell Culture

• Before adding to cell cultures, dilute the stock solution to the desired working concentration using cell culture medium or PBS containing 0.1% BSA or 10% FBS as a carrier.
• Filter-sterilize the final working solution if sterility is required and the protein is not already sterile-filtered.

5. General Handling Tips

• Mix all solutions gently and thoroughly.
• Avoid prolonged exposure to room temperature.
• Always consult the specific product datasheet for any unique instructions, as formulations may vary between suppliers.

Example Protocol

1. Equilibrate the lyophilized sTNF RI vial to room temperature.2. Add sterile PBS (with 0.1% BSA if carrier-free) to achieve 100 μg/mL.3. Gently swirl to dissolve; incubate at room temperature for 15–30 minutes.4. Aliquot and store at –20°C or –70°C.5. For experiments, dilute aliquots to working concentration in cell culture medium with carrier protein.

Key Points

• Sterile PBS (with or without 0.1% BSA) is the standard reconstitution buffer.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Dilute in cell culture medium with carrier protein for use in experiments.

These steps ensure optimal protein stability and activity for cell culture applications.