Recombinant Human CD120b (TNFR2)

Recombinant Human CD120b (TNFR2) is a valuable tool in research applications due to its central role in immune regulation, cell signaling, and disease pathogenesis, particularly in cancer, autoimmunity, and inflammation.

Key scientific reasons to use recombinant human CD120b (TNFR2):

• Immune Regulation: TNFR2 (CD120b) is highly expressed on activated T and B lymphocytes, especially regulatory T cells (Tregs), where it is crucial for their proliferation, stability, and suppressive function. Manipulating TNFR2 signaling allows researchers to study Treg biology and immune tolerance mechanisms.

• Cancer Immunology: TNFR2 is a promising target for cancer immunotherapy. It is upregulated on Tregs within the tumor microenvironment, contributing to immunosuppression and tumor progression. Recombinant TNFR2 can be used to investigate checkpoint inhibition strategies, Treg depletion, and the enhancement of anti-tumor immune responses.

• Inflammation and Autoimmunity: Sustained TNFR2 signaling is implicated in the pathogenesis of diseases such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. Recombinant TNFR2 enables mechanistic studies of cytokine signaling and the development of therapeutic agents.

• Decoy Receptor Function: The soluble extracellular domain of TNFR2 acts as a decoy receptor, binding TNF-α and inhibiting its pro-inflammatory effects. Recombinant soluble TNFR2 is used to study cytokine neutralization and as a model for therapeutic agents.

• Cell Signaling Pathways: TNFR2 engagement activates NF-κB and mTOR pathways, influencing cell survival, proliferation, and metabolism. Recombinant TNFR2 is essential for dissecting these signaling cascades in vitro.

Typical research applications include:

• Functional assays to study Treg expansion, stability, and suppressive activity.
• Investigating TNFR2-mediated signaling in immune and non-immune cells.
• Screening and characterization of TNFR2-targeting antibodies or small molecules.
• Modeling cytokine-receptor interactions and downstream effects.
• Developing and validating immunotherapeutic strategies targeting TNFR2.

Best practices:

• Use recombinant TNFR2 in controlled in vitro assays to ensure specificity and reproducibility.
• Select the appropriate isoform (full-length, extracellular domain, or Fc-fusion) based on experimental needs.
• Confirm purity and biological activity using SDS-PAGE, endotoxin testing, and functional assays.

In summary, recombinant human CD120b (TNFR2) is indispensable for dissecting immune mechanisms, modeling disease processes, and developing novel therapeutics targeting TNF signaling pathways.

You can use recombinant human CD120b (TNFR2) as a standard for quantification or calibration in your ELISA assays, provided that the protein is of sufficient purity, its concentration is accurately determined, and it is compatible with your assay format.

Key considerations:

• Purity and Quantification: For ELISA standard curves, it is essential to use a highly purified protein or a recombinant protein whose concentration has been precisely measured, ideally by methods such as HPLC or absorbance at 280 nm. Impurities or inaccurate quantification can lead to unreliable calibration.

• Form and Compatibility: The recombinant TNFR2 should be in a form (e.g., soluble, properly folded, and free of tags or modifications that interfere with antibody binding) that is recognized by the capture and detection antibodies used in your ELISA. If your ELISA is designed to detect soluble TNFR2, ensure your recombinant standard matches this form.

• Validation: If you are developing or modifying an ELISA, you should validate the use of your recombinant TNFR2 as a standard by confirming linearity, accuracy, and parallelism with native samples. This ensures that the recombinant protein behaves similarly to endogenous TNFR2 in your assay matrix.

• Concentration Range: Prepare a standard curve covering the expected concentration range in your samples. Commercial ELISA kits for soluble TNFR2 typically use standard curves in the range of 0.1–142 ng/mL.

Best Practices:

• Reconstitute or dilute the recombinant TNFR2 in the same buffer or matrix as your samples to minimize matrix effects.
• Store aliquots of the standard at recommended conditions to avoid degradation or repeated freeze-thaw cycles.
• Validate each new lot of recombinant protein for consistency.

Summary Table: Requirements for ELISA Standard

If your recombinant human CD120b (TNFR2) meets these criteria, it is suitable for use as a standard in ELISA quantification and calibration.

Recombinant human CD120b (TNFR2) has been validated for a diverse range of applications in published research, spanning both analytical and functional studies.

Analytical Applications

The recombinant protein has been extensively validated for standard biochemical and immunological techniques. Western blotting represents one of the primary applications, with successful detection demonstrated in multiple cell lines including U-937, K-562, THP-1, NK-92, Jurkat, and HEK-293 cells, as well as in thymus tissue from human, mouse, and rat sources. ELISA applications have also been well-established, with the recombinant protein serving effectively in both sandwich and capture formats. Immunohistochemistry (IHC) has been validated using the recombinant protein, particularly for detecting CD120b expression in human spleen tissue.

Additional analytical techniques validated include immunofluorescence/immunocytochemistry (IF/ICC) in cell culture systems, immunoprecipitation (IP) in HEK-293 cells, and co-immunoprecipitation (CoIP) applications. Flow cytometry represents another validated application for this recombinant protein.

Functional Applications

Beyond analytical uses, recombinant CD120b has been validated in functional assays designed to assess receptor-ligand interactions and signaling capacity. The protein has demonstrated utility in blocking studies, where it serves to antagonize TNF-α and lymphotoxin-alpha (LT-α) signaling.

Research Context

In published cancer immunotherapy research, anti-TNFR2 antagonistic approaches have been validated in both in vitro and in vivo models, demonstrating therapeutic potential through inhibition of regulatory T cell proliferation and enhancement of antitumor immunity. These applications underscore the recombinant protein's relevance for investigating TNF receptor signaling in disease pathogenesis and therapeutic intervention strategies.

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

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Reconstitute the protein in sterile distilled water or sterile 18 MΩ-cm H₂O. The recommended concentration for reconstitution is typically 0.1–0.5 mg/mL (100–500 µg/mL), but not less than 100 µg/mL. For example, to prepare a 0.5 mg/mL solution, add 200 µL water to 100 µg lyophilized protein.
• Gently mix by swirling or inverting; avoid vortexing or vigorous pipetting to prevent protein denaturation.
• If the protein was lyophilized from PBS (pH 7.4) with stabilizers (e.g., trehalose), reconstitution in water is sufficient. If not, check the datasheet for specific buffer requirements.
• Optional: For long-term stability, add a carrier protein or stabilizer (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) after reconstitution, especially if you plan to store aliquots.
• Aliquot the reconstituted solution to minimize freeze-thaw cycles.
• Storage after reconstitution:
  • Short-term: 2–8 °C for up to 1 week.
  • Long-term: –20 °C or below for up to 3 months; avoid repeated freeze-thaw cycles.
• Working solution: Dilute the stock to the desired concentration in cell culture medium immediately before use. Ensure the final buffer is compatible with your cells (e.g., isotonic, serum-free if required).

Additional notes:

• Always consult the specific product datasheet for any unique instructions, as formulations and stabilizers may vary between preparations.
• If the protein is to be used in functional assays (e.g., TNF-α inhibition), confirm activity with a pilot experiment, as some proteins may require gentle handling or specific buffers for optimal function.

Summary of key steps:

• Centrifuge vial → Add sterile water (0.1–0.5 mg/mL) → Gently mix → Add stabilizer if needed → Aliquot → Store appropriately → Dilute for cell culture use.

This protocol ensures maximal protein stability and activity for cell culture applications.