Recombinant Mouse CD120b (TNFR2)

Recombinant Mouse CD120b (TNFR2) is widely used in research because it is a key receptor for TNF-α and plays a central role in immune regulation, inflammation, cell proliferation, apoptosis, and cancer immunology. Using the recombinant form allows for precise, reproducible studies of TNFR2 function and its interactions in various biological systems.

Key reasons to use recombinant mouse CD120b (TNFR2) in research applications:

• Mechanistic Studies: TNFR2 is a high-affinity receptor for TNF-α and is involved in signaling pathways that regulate cell proliferation, differentiation, apoptosis, and immune responses. Recombinant TNFR2 enables controlled in vitro and in vivo experiments to dissect these pathways.
• Immunology and Cancer Research: TNFR2 is highly expressed on regulatory T cells (Tregs), where it is crucial for their suppressive function and stability. Targeting TNFR2 can modulate Treg activity, making it a promising target for cancer immunotherapy and studies of immune tolerance.
• Decoy Receptor Function: The soluble recombinant form of TNFR2 can act as a decoy receptor, binding TNF-α and inhibiting its activity, which is useful for studying TNF-α-mediated cytotoxicity and inflammation.
• Assay Standardization: Recombinant TNFR2 is used as a standard in ELISA and other binding assays to quantify TNFR2 levels or to test the activity of antibodies and ligands targeting TNFR2.
• Therapeutic Target Validation: Recombinant TNFR2 is essential for screening and characterizing antibodies or small molecules that block or activate TNFR2, supporting drug discovery and validation efforts.

Additional technical considerations:

• Recombinant mouse CD120b (TNFR2) is typically produced with high purity and low endotoxin levels, ensuring suitability for sensitive cell-based assays and in vivo studies.
• The protein is often supplied as the extracellular domain, which is sufficient for ligand binding and functional studies.

In summary, recombinant mouse CD120b (TNFR2) is a versatile tool for dissecting TNF signaling, studying immune regulation, developing immunotherapies, and standardizing bioassays in mouse models.

You can use recombinant Mouse CD120b (TNFR2) as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant proteins are commonly used as standards in quantitative ELISA, but several important considerations apply.

Key points and supporting details:

• Recombinant proteins as ELISA standards: It is standard practice to use a purified recombinant protein as the calibrator for quantitative ELISA assays, especially when the native protein is difficult to obtain or purify in sufficient quantities. Many commercial ELISA kits for mouse TNFR2 (CD120b) explicitly include a recombinant protein as the standard.

• Validation and compatibility: Not all ELISA kits are validated for recombinant standards. Some kits are designed to detect only the native form of the protein and may not recognize recombinant forms due to differences in folding, glycosylation, or other post-translational modifications. For example, certain kits specify: "The ELISA Kit is designed to detect native, not recombinant, TNFRSF1B". In such cases, using a recombinant standard may result in inaccurate quantification.

• Best practices:

  • Confirm that your ELISA antibodies recognize the recombinant form of CD120b (TNFR2) with the same affinity as the native protein.
  • Prepare the standard curve using the recombinant protein at known concentrations, ideally in the same buffer or matrix as your samples to minimize matrix effects.
  • Validate the linearity and parallelism of the standard curve with your sample matrix to ensure accurate quantification.
  • If using a commercial kit, check the kit documentation or contact technical support to confirm compatibility with recombinant standards.

• Typical use in commercial kits: Many quantitative mouse TNFR2 ELISA kits provide a lyophilized recombinant mouse TNFR2 standard for calibration, with standard curves typically ranging from 9.4–1000 pg/ml. This demonstrates that recombinant TNFR2 is widely accepted as a standard when properly validated.

Summary Table: Recombinant Mouse CD120b (TNFR2) as ELISA Standard

Conclusion:
You can use recombinant Mouse CD120b (TNFR2) as a standard for ELISA quantification if your assay is validated for it and the antibodies recognize the recombinant form equivalently to the native protein. Always check your kit's documentation or validate in-house to ensure accurate results.

Recombinant Mouse CD120b (TNFR2) has been validated for a range of applications in published research, primarily as a functional ligand or decoy receptor in both in vitro and in vivo studies. The most common validated applications include:

• In vitro functional assays: Recombinant mouse TNFR2 is widely used to study TNF-α signaling, often by acting as a decoy receptor to inhibit TNF-α–mediated cytotoxicity in cell-based assays (e.g., using mouse L-929 cells). This allows researchers to dissect the specific contributions of TNFR2 signaling in processes such as cell proliferation, differentiation, apoptosis, and immune regulation.

• In vivo blockade studies: Recombinant TNFR2, or antibodies targeting TNFR2, are used in animal models to block TNFR2 signaling, enabling the study of its role in immune responses, inflammation, tumor growth, and autoimmune diseases. These studies often involve administration of recombinant protein or blocking antibodies to mice and subsequent analysis of physiological or pathological outcomes.

• Receptor-ligand binding studies: Recombinant TNFR2 is used in biochemical assays to characterize binding interactions with its ligands (TNF-α, LT-α) or to screen/select for TNFR2-selective agonists or antagonists. For example, phage display and other binding assays utilize recombinant TNFR2 to identify molecules that specifically interact with the receptor.

• ELISA and immunoassays: Recombinant TNFR2 serves as a standard or capture antigen in ELISA to quantify TNFR2 or its ligands in biological samples. It is also used to validate antibody specificity in immunoassays.

• Flow cytometry and immunoprecipitation: While recombinant TNFR2 itself is not directly used for flow cytometry, it is employed to validate antibody specificity or as a coating antigen for bead-based assays. It is also used in immunoprecipitation protocols to pull down interacting proteins or antibodies.

• Cellular differentiation and signaling studies: Recombinant TNFR2 is used to stimulate or modulate T cell subsets, particularly regulatory T cells (Tregs), to study their differentiation, migration, and immunosuppressive functions in vitro and in vivo. These studies have implications for autoimmune disease, transplantation, and cancer immunology.

Summary Table of Validated Applications

Key details:

• Recombinant mouse TNFR2 is most often used as a soluble extracellular domain, acting as a decoy receptor or functional ligand in these assays.
• Its biological activity is typically validated by its ability to inhibit TNF-α–mediated cytotoxicity in cell lines such as L-929.
• Applications are supported by both commercial validation and peer-reviewed research, particularly in immunology, oncology, and inflammation studies.

If you require protocols or more specific examples from primary literature, please specify the application of interest.

To reconstitute and prepare Recombinant Mouse CD120b (TNFR2) protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly to ensure all lyophilized powder is at the bottom before opening.
• Reconstitute the protein in sterile water or sterile 18 MΩ-cm H₂O. A typical concentration range is 0.1–0.5 mg/mL. For example, if you have 100 µg of protein, add 200–1000 µL of sterile water to achieve a final concentration between 0.1 and 0.5 mg/mL.
• Gently mix by pipetting up and down or swirling; avoid vigorous vortexing to prevent protein denaturation.
• Let the solution sit at room temperature for 5–10 minutes to ensure complete dissolution.

Storage after reconstitution:

• Short-term: Store at 4°C for up to one week.
• Long-term: Aliquot into working volumes to avoid repeated freeze-thaw cycles and store at –20°C or –80°C.

Dilution for cell culture:

• After reconstitution, dilute the stock solution into your desired cell culture medium (e.g., DMEM, RPMI) to the working concentration required for your experiment. This is typically in the range of ng/mL to low µg/mL, depending on the biological assay and cell type.
• If the protein was lyophilized with additives (e.g., acetonitrile, TFA), ensure the final concentration of these solvents in your culture is negligible by sufficient dilution, or consider buffer exchange if necessary.

General notes:

• Use sterile technique throughout to prevent contamination.
• If the protein is sensitive to oxidation or aggregation, consider adding a carrier protein (e.g., 0.1% BSA) to the reconstitution buffer, unless this interferes with your assay.
• Always consult the specific product datasheet for any unique instructions regarding reconstitution or storage, as formulations may vary.

Summary protocol:

1. Centrifuge vial, add sterile water to 0.1–0.5 mg/mL, mix gently, let dissolve.
2. Aliquot and store at –20°C or –80°C for long-term use.
3. Dilute into cell culture medium to desired working concentration immediately before use.

These steps will ensure the Recombinant Mouse CD120b (TNFR2) protein is properly prepared for reliable cell culture experiments.