Recombinant Human TNFRSF14

Recombinant Human TNFRSF14 (also known as HVEM or CD270) is a versatile immunoregulatory protein that can be used in research to study T cell activation, immune modulation, tumor immunity, autoimmune disease mechanisms, and cell signaling pathways. Its recombinant form enables controlled, reproducible experiments in vitro and in vivo.

Key scientific applications and rationale for using recombinant TNFRSF14 include:

• T Cell Activation and Proliferation: TNFRSF14 acts as a co-stimulatory molecule for T cells. When engaged by its ligand LIGHT (TNFSF14), it stimulates T cell proliferation and cytokine production, which is crucial for studying cellular immune responses, inflammation, and CTL-mediated tumor immunity.

• Tumor Immunity and Cancer Research: The LIGHT-HVEM pathway enhances anti-tumor immune responses, including cytotoxic T lymphocyte (CTL) activation and tumor cell apoptosis. Recombinant TNFRSF14 can be used to dissect these mechanisms and evaluate immunotherapeutic strategies.

• Autoimmune and Transplantation Models: TNFRSF14 is implicated in the regulation of autoimmune diseases and transplant rejection. Its recombinant form allows for the investigation of co-stimulatory and inhibitory signals in T and B cells, helping to elucidate the pathogenesis of conditions such as autoimmune diabetes, hepatitis, and rheumatoid arthritis.

• Functional Assays and Blocking Studies: Recombinant TNFRSF14 is validated for use in blocking assays and functional assays, enabling precise modulation of immune pathways in experimental systems.

• Cell Differentiation and Survival: The LIGHT-TNFRSF14 axis promotes differentiation and survival of various cell types, including bone marrow-derived mesenchymal stem cells (hBM-MSCs) into hepatocyte-like cells, making it valuable for stem cell and regenerative medicine research.

• Infection and Antibacterial Immunity: TNFRSF14 plays a role in the immune response to viral (e.g., HSV) and bacterial infections by enhancing phagocytosis and inflammatory responses.

• Asthma and Allergy Models: TNFRSF14 expression on mast cells contributes to the development of asthma pathology, including airway inflammation and hyperresponsiveness, making recombinant TNFRSF14 useful for studying allergic disease mechanisms.

• Memory T Cell and Treg Function: HVEM signaling is involved in the development of CD8+ T cell memory and regulatory T cell (Treg) function, relevant for immunological tolerance and homeostasis studies.

Best Practices:

• Use recombinant TNFRSF14 in controlled dose-response experiments to analyze its effects on immune cell activation, differentiation, or apoptosis.
• Employ it in co-culture systems with its ligands (e.g., LIGHT) to dissect bidirectional signaling and immune modulation.
• Apply blocking or functional assays to investigate the role of TNFRSF14 in specific disease models or signaling pathways.

Summary:
Recombinant Human TNFRSF14 is a powerful tool for immunology, oncology, stem cell biology, and infectious disease research, enabling mechanistic studies of immune regulation, cell signaling, and therapeutic intervention strategies.

Yes, you can use Recombinant Human TNFRSF14 as a standard for quantification or calibration in your ELISA assays, provided it is suitable for the specific assay format and detection system you are using.

Recombinant Human TNFRSF14 (also known as HVEM) is commonly used as a reference standard in ELISA kits to generate a standard curve for quantifying the target protein in samples. For example, several commercial ELISA kits for TNFRSF14 (such as those from R&D Systems, Thermo Fisher Scientific, and others) include recombinant TNFRSF14 as part of their standard curve. The standard curve allows you to interpolate the concentration of TNFRSF14 in your samples based on the signal generated.

Key Points:

• Recombinant TNFRSF14 is typically provided as a purified protein (often as an Fc chimera or in another fusion format) and is suitable for use as a standard in sandwich ELISA assays.
• Ensure that the recombinant protein is compatible with the antibodies used in your ELISA (i.e., the epitopes recognized by the capture and detection antibodies should be present in the recombinant standard).
• The formulation of the recombinant protein (e.g., with or without BSA, buffer composition) may affect its performance as a standard, so it is important to follow the manufacturer's recommendations.
• If you are developing your own ELISA, you may need to optimize the concentration range of the recombinant standard to match the expected levels in your samples.

In summary, recombinant Human TNFRSF14 is a valid and commonly used standard for calibration in ELISA assays targeting TNFRSF14.

Recombinant Human TNFRSF14 (also known as HVEM or CD270) has been validated for several applications in published research, primarily in the context of immunology, cell signaling, and protein-protein interaction studies.

Key validated applications include:

• Functional Assays: Used to study the biological activity of TNFRSF14, such as its ability to deliver costimulatory signals to T cells, promote T cell proliferation, and enhance cytokine production. Functional assays also include its role in immune cell survival, differentiation, and effector functions.
• Blocking Assays: Applied to investigate the inhibition of TNFRSF14-mediated signaling pathways, often to dissect its role in immune modulation.
• Binding Assays/Protein-Protein Interaction: Used to characterize interactions between TNFRSF14 and its ligands (e.g., TNFSF14/LIGHT, BTLA, CD160), often via ELISA or similar platforms. These assays confirm the protein’s binding specificity and affinity.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized as a standard or analyte to quantify protein-protein interactions or ligand binding.
• SDS-PAGE: Used for protein characterization and purity assessment.
• Western Blot (WB): Sometimes used as a positive control for antibody validation or detection of TNFRSF14 in samples.

Research applications and biological contexts:

• Immune modulation: TNFRSF14 is widely used to study T cell costimulation, immune checkpoint regulation, and the balance between immune activation and inhibition.
• Cancer immunotherapy: Recombinant TNFRSF14 has been used in preclinical models to investigate its role in enhancing anti-tumor immune responses, often in combination with other immunotherapies.
• Stem cell biology: Recombinant TNFSF14 (the ligand for TNFRSF14) has been used to modulate mesenchymal stem cell (MSC) proliferation and survival, suggesting potential applications in regenerative medicine.
• Infection and inflammation: TNFRSF14’s role as a receptor for viral entry (e.g., Herpes simplex virus) and in the regulation of antimicrobial and pro-inflammatory responses has been explored.

Summary Table of Validated Applications

Note: The specific application and validation may depend on the recombinant protein’s format (e.g., tag, expression system) and the experimental context. Always consult the primary literature or product datasheets for detailed protocols and validation data relevant to your research needs.

To reconstitute and prepare Recombinant Human TNFRSF14 protein for cell culture experiments, dissolve the lyophilized protein in sterile, endotoxin-free water or PBS to a concentration between 0.1–1.0 mg/mL, depending on your experimental requirements. For optimal stability and activity in cell culture, consider the following protocol:

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Add sterile, endotoxin-free water or PBS to the vial. Common reconstitution concentrations are:
   • 0.1–1.0 mg/mL for general use.
   • 100–500 μg/mL for specific bioassays or ELISA standards.
3. Optional: Add carrier protein or stabilizer (if not already present in the formulation):
   • Add 0.1%–1% bovine serum albumin (BSA) or human serum albumin to prevent adsorption and enhance stability, especially for cell culture applications.
   • Alternatively, add 5–50% glycerol for long-term storage and freeze-thaw protection.
4. Mix gently by pipetting up and down or by gentle vortexing. Avoid vigorous agitation to prevent protein denaturation.
5. Allow the protein to dissolve completely (typically 5–10 minutes at room temperature). Inspect for complete dissolution; if necessary, incubate briefly at room temperature.
6. Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
7. Storage:
   • Store aliquots at –20°C or below for long-term use.
   • For short-term use (2–7 days), store at 4°C.
   • Avoid repeated freeze-thaw cycles to maintain protein activity.

Additional notes for cell culture:

• Ensure the final buffer is compatible with your cell culture system (e.g., PBS, Tris-buffered saline, or cell culture medium).
• Confirm endotoxin levels are below 1 EU/μg for sensitive cell types.
• If using a carrier-free formulation, consider adding BSA if protein loss or instability is observed.
• Filter-sterilize the final solution if sterility is required and not guaranteed by the manufacturer.

Example preparation for cell culture:

- Reconstitute lyophilized TNFRSF14 in sterile PBS containing 0.1% BSA to 100 μg/mL.- Aliquot and store at –20°C.- Thaw aliquots on ice before use and dilute to working concentration in cell culture medium.

Summary of key points:

• Reconstitute in sterile water or PBS to 0.1–1.0 mg/mL.
• Add carrier protein or glycerol if needed for stability.
• Aliquot and store at –20°C or below; short-term at 4°C.
• Avoid repeated freeze-thaw cycles.

Always consult the specific product datasheet for formulation details and recommended reconstitution buffer, as some preparations may contain stabilizers (e.g., trehalose, glycine, Tris).