Recombinant Human Lymphotoxin βR

Recombinant Human Lymphotoxin βR (LTβR) is a valuable tool in research applications focused on immunology, inflammation, cancer, and tissue homeostasis because it enables precise investigation of LTβR-mediated signaling pathways and cellular responses.

Key scientific applications and rationale:

• Dissecting LTβR Signaling: LTβR is a critical receptor in the TNF superfamily, regulating the development and organization of lymphoid organs, mucosal immune responses, and homeostasis of immune cell populations such as neutrophils, NK, and iNKT cells. Using recombinant LTβR allows researchers to study ligand-receptor interactions, downstream signaling (e.g., NF-κB activation), and the effects on immune cell function in controlled in vitro or in vivo systems.

• Modeling Disease Mechanisms: LTβR signaling is implicated in autoimmune diseases, infectious diseases, cancer, and renal injury. Recombinant LTβR can be used in bioassays to model disease-relevant pathways, test therapeutic interventions, or evaluate the impact of LTβR modulation on inflammation, tissue regeneration, and carcinogenesis.

• Cellular and Molecular Assays: Recombinant LTβR is suitable for applications such as flow cytometry, bioassays, and surface plasmon resonance to quantify ligand binding, receptor activation, and downstream effects (e.g., cytokine and chemokine production, adhesion molecule expression). This is essential for mechanistic studies and drug screening.

• Functional Studies: LTβR engagement induces production of key molecules (IL-7, RANKL, VEGF-C, VCAM-1, ICAM-1, MAdCAM, CXCL13, CCL19, CCL21) that regulate immune cell trafficking, lymphoid tissue architecture, and inflammatory responses. Recombinant LTβR enables functional studies of these processes in human cell systems.

• Translational Research: Because LTβR is upregulated in conditions such as hepatitis, hepatocellular carcinoma, and atherosclerosis, recombinant human LTβR provides a human-relevant model for translational studies, including therapeutic antibody development and biomarker discovery.

Best practices:

• Use recombinant LTβR in species-matched systems (e.g., human cells) to ensure physiological relevance.
• Validate receptor functionality and ligand specificity in your assay system.
• Employ appropriate controls (e.g., blocking antibodies, ligand mutants) to confirm LTβR-dependent effects.

Summary of advantages:

• Enables mechanistic dissection of LTβR pathways in human cells.
• Facilitates disease modeling and therapeutic screening.
• Supports functional and translational studies in immunology, oncology, and tissue biology.

In summary, recombinant human LTβR is essential for elucidating the biological roles and therapeutic potential of LTβR signaling in human health and disease.

You can use recombinant human Lymphotoxin βR as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity and its concentration is accurately known. This is a common practice in quantitative ELISA, where a standard curve is generated using known concentrations of a purified or recombinant protein to determine the concentration of the target analyte in your samples.

Key considerations:

• Purity and Characterization: The recombinant protein should be well-characterized and as pure as possible to ensure accurate quantification.
• Standard Curve Preparation: Prepare a standard curve by serially diluting the recombinant protein in the same buffer or matrix as your samples, covering the expected concentration range of your assay.
• Matrix Effects: If your samples are in a complex matrix (e.g., serum, plasma), consider diluting your standard in the same matrix to minimize matrix effects and improve accuracy.
• Antibody Recognition: The recombinant standard must contain the same epitopes recognized by the capture and detection antibodies in your ELISA. If the recombinant protein is truncated, tagged, or otherwise modified, confirm that it is fully recognized by your assay antibodies.
• Lot-to-Lot Variability: Be aware that different lots of recombinant protein may have slight differences in immunoreactivity or quantification, so it is best to value-assign the standard concentration based on measurement in your ELISA rather than relying solely on the mass stated on the vial.
• Dilution Accuracy: Large dilutions from stock concentrations (e.g., µg/mL to pg/mL) can introduce pipetting errors, so use calibrated pipettes and prepare intermediate dilutions as needed.

Protocol summary:

1. Reconstitute the recombinant Lymphotoxin βR according to the supplier’s instructions.
2. Prepare a series of dilutions to generate a standard curve (e.g., 0–1000 pg/mL or as appropriate for your assay’s sensitivity and dynamic range).
3. Run the ELISA with your standards and samples in parallel.
4. Use the standard curve to interpolate the concentrations of Lymphotoxin βR in your samples.

Limitations:

• If your ELISA kit is calibrated against a different standard (e.g., a native protein or a specific recombinant isoform), results may not be directly comparable unless you validate the equivalence of your recombinant standard.
• Always check the ELISA kit documentation for any specific recommendations or restrictions regarding the use of external standards.

In summary, recombinant human Lymphotoxin βR is suitable as a standard for ELISA quantification if it is pure, well-characterized, and recognized by your assay antibodies, and if you carefully prepare and validate your standard curve.

Recombinant Human Lymphotoxin βR (LTβR) has been validated for several applications in published research, primarily as a tool to study LTβR signaling, immune modulation, and ligand interactions.

Validated Applications in Published Research:

• Bioassays:
  Recombinant LTβR is widely used in cell-based bioassays to study its role in signaling pathways, such as the activation of NF-κB, induction of cytokines, and regulation of immune responses in various cell types, including hepatocytes, stromal cells, and melanoma cells. It has been used to demonstrate its influence on inflammation, lymphoid organogenesis, and carcinogenesis.

• Flow Cytometry:
  The recombinant protein has been validated for use in flow cytometry to detect LTβR expression on the surface of human cells, particularly stromal and epithelial cells, but not lymphocytes.

• Surface Plasmon Resonance (SPR):
  SPR has been employed to characterize the binding kinetics and affinity of LTβR with its ligands, such as LIGHT and lymphotoxin α1β2, and to evaluate engineered ligand variants for therapeutic applications.

• Blocking/Decoy Receptor Studies:
  Soluble recombinant LTβR (often as an Fc fusion protein) is used as a decoy receptor to inhibit endogenous LTβR signaling in vitro and in vivo. This approach has been instrumental in dissecting the role of LTβR in autoimmune disease models, lymphoid tissue organization, and inflammatory responses.

• Tumor Cell Cytotoxicity Assays:
  Recombinant LTβR fusion proteins are used in cytotoxicity assays to monitor LTβR-mediated signaling and screen for potential therapeutic agents that modulate this pathway.

Additional Context:

• Sample Types:
  Applications have been validated on whole cells, including primary human cells and established cell lines.

• Research Areas:
  Studies have focused on immunology (autoimmunity, lymphoid organ development, mucosal immunity), oncology (tumor microenvironment, cancer therapy), and inflammation (rheumatoid arthritis, hepatitis).

• Mechanistic Studies:
  Recombinant LTβR has been used to elucidate downstream signaling mechanisms, such as canonical and alternative NF-κB activation, and to study the induction of chemokines, adhesion molecules, and cytokines.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are central to research on LTβR biology and therapeutic targeting.

To reconstitute and prepare Recombinant Human Lymphotoxin βR protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS, pH 7.4) to a concentration between 0.1–1.0 mg/mL, avoiding vigorous mixing or vortexing. Use gentle pipetting or slow agitation to facilitate dissolution, and allow the protein to fully dissolve at room temperature for 15–30 minutes.

Key steps and best practices:

• Centrifuge the vial briefly (3000–3500 rpm, 5 min) before opening to collect all powder at the bottom.
• Dissolve in sterile PBS (pH 7.4); if recommended by your datasheet, include a carrier protein such as 0.1%–1% BSA, HSA, or FBS to stabilize the protein and prevent adsorption to tube walls.
• Do not vortex; instead, gently pipette or swirl to mix.
• Incubate at room temperature for 15–30 minutes to ensure complete dissolution.
• Dilute to working concentration using cell culture medium or PBS containing carrier protein as needed for your experiment.
• Aliquot the solution to avoid repeated freeze-thaw cycles, which can reduce protein activity.
• Storage after reconstitution:
  • Short-term: 2–8 °C for up to 1 month.
  • Long-term: –20 °C to –70 °C, preferably in aliquots.
  • Avoid repeated freeze-thaw cycles.

Additional notes:

• If your experiment requires serum-free conditions, use a non-protein stabilizer such as trehalose instead of BSA or FBS.
• For cell culture, add the reconstituted protein directly to the medium at the desired final concentration, typically in the range of 0.02–0.1 μg/mL for bioassays, but refer to your specific protocol or datasheet for optimal dosing.
• Always consult the product’s Certificate of Analysis (CoA) or datasheet for any manufacturer-specific recommendations.

Summary protocol:

1. Centrifuge vial, open carefully.
2. Add sterile PBS (pH 7.4) with 0.1%–1% BSA to achieve 0.1–1.0 mg/mL.
3. Mix gently, incubate at room temperature 15–30 min.
4. Dilute as needed with carrier protein-containing buffer.
5. Aliquot and store at 2–8 °C (short-term) or –20 °C to –70 °C (long-term).
6. Avoid repeated freeze-thaw cycles.

This protocol ensures maximal stability and bioactivity of recombinant Lymphotoxin βR for cell culture applications.