Recombinant Human LIGHT

Recombinant Human LIGHT (TNFSF14) is a valuable tool for a wide range of research applications due to its well-characterized biological functions and reproducible activity. Here are key reasons to use Recombinant Human LIGHT in your research:

1. Consistent and Reproducible Activity

Recombinant Human LIGHT is produced under controlled conditions, ensuring batch-to-batch consistency and high purity. This reproducibility is essential for reliable and interpretable experimental results, especially in quantitative assays and dose-response studies.

2. Well-Defined Biological Functions

LIGHT (TNFSF14) is a member of the tumor necrosis factor (TNF) superfamily and plays critical roles in:

• Immune regulation: LIGHT binds to receptors such as HVEM, LTβR, and DcR3, modulating immune cell activation, proliferation, and apoptosis.
• Cell proliferation and survival: It stimulates proliferation in human umbilical vein endothelial cells (HUVECs) and enhances the survival and proliferation of human bone marrow-derived mesenchymal stem cells.
• Apoptosis induction: LIGHT overexpression in tumor cells induces apoptosis, which can be further enhanced by IFN-γ.
• Tissue differentiation: LIGHT promotes the differentiation of mesenchymal stem cells into functional hepatocyte-like cells and enhances osteogenesis.

3. Broad Applicability in Disease Models

Recombinant Human LIGHT is used in studies of:

• Inflammation and autoimmune diseases: LIGHT is upregulated in conditions such as rheumatoid arthritis and coronary disease, where it mediates inflammatory responses and cellular adhesion.
• Cancer research: LIGHT induces apoptosis in certain tumor cells and can be used to study tumor-immune interactions.
• Metabolic and cardiovascular diseases: LIGHT regulates inflammatory responses and lipid metabolism in macrophages, making it relevant for studies of atherosclerosis and metabolic syndrome.
• Tissue engineering and regenerative medicine: LIGHT supports stem cell survival, proliferation, and differentiation, which are crucial for tissue regeneration.

4. Compatibility with Multiple Assay Types

Recombinant Human LIGHT is suitable for a variety of experimental applications, including:

• Bioassays: To study cell proliferation, apoptosis, and differentiation.
• Surface Plasmon Resonance (SPR): To investigate receptor-ligand interactions.
• In vitro and in vivo models: For mechanistic studies and therapeutic development.

5. Support for Mechanistic Studies

Using recombinant LIGHT allows researchers to dissect specific signaling pathways, such as NF-κB activation, and to explore the roles of LIGHT in cellular processes like adhesion, migration, and matrix production.

6. Clinical and Translational Relevance

Findings from studies using recombinant LIGHT have direct implications for understanding disease mechanisms and developing targeted therapies. For example, LIGHT’s role in immune regulation and tissue repair makes it a candidate for therapeutic intervention in autoimmune, inflammatory, and degenerative diseases.

7. Availability and Quality

Recombinant Human LIGHT is available from multiple reputable suppliers, ensuring access to high-quality, well-characterized protein for research use.

In summary, Recombinant Human LIGHT is a versatile and reliable reagent for studying immune regulation, cell survival, differentiation, and disease mechanisms, making it an essential tool for researchers in immunology, oncology, regenerative medicine, and related fields.

Yes, you can use recombinant human LIGHT as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately determined. Recombinant proteins are commonly used as standards in quantitative ELISA protocols, especially when natural protein is scarce or difficult to purify.

Key considerations and best practices:

• Purity and Quantification: The recombinant human LIGHT should be highly purified. Its concentration should be determined by reliable methods such as HPLC, UV spectrophotometry, or amino acid analysis to ensure accurate calibration.
• Parallelism: The standard curve generated using recombinant human LIGHT should be validated for parallelism with curves generated from natural human LIGHT. This ensures that the assay quantifies both forms equivalently. Commercial ELISA kits for human LIGHT report that their recombinant standards produce parallel curves with natural LIGHT, supporting their use for quantification.
• Matrix Effects: When using recombinant LIGHT as a standard, ensure that the diluent and matrix used for the standard curve closely match those of your samples to minimize matrix effects and improve accuracy.
• Lot Consistency: Use standards from the same lot throughout your experiments, as calibrators can be lot-specific and may vary between batches.
• Validation: If you are developing your own ELISA, validate the recombinant standard by spiking known amounts into sample matrices and assessing recovery, linearity, and precision.

Limitations:

• Research Use Only: Most recombinant standards and ELISA kits are for research use only and not for diagnostic procedures.
• Bioactivity vs. Immunoreactivity: Recombinant proteins used as ELISA standards should be validated for immunoreactivity, not just bioactivity. Some recombinant preparations may be suitable for bioassays but not for ELISA calibration unless specifically tested for this purpose.

Protocol summary for using recombinant human LIGHT as an ELISA standard:

1. Prepare serial dilutions of the recombinant human LIGHT in the same buffer or matrix as your samples.
2. Run these standards in parallel with your samples on the ELISA plate.
3. Generate a standard curve by plotting absorbance versus concentration.
4. Use the curve to interpolate the concentration of LIGHT in your samples.

In summary: Recombinant human LIGHT is suitable as a standard for ELISA quantification if it is highly purified, accurately quantified, and validated for immunoreactivity in your assay system. Always confirm parallelism and recovery to ensure reliable quantification.

Recombinant Human LIGHT (TNFSF14) has been validated for several key applications in published research, primarily in bioassays, cell culture studies, and biophysical interaction analyses.

Validated Applications:

• Bioassays:
  Recombinant Human LIGHT is extensively used in bioassays to study its biological activity, such as stimulating cell proliferation (e.g., HUVEC endothelial cells), inducing apoptosis in tumor cells, and modulating inflammatory responses in various cell types including macrophages, monocytes, and synoviocytes.
  Examples include:

  • Induction of apoptosis in tumor cells (often enhanced by IFN-γ).
  • Regulation of inflammatory response and lipid metabolism in macrophages.
  • Promotion of keratinocyte activity and skin fibrosis.
  • Activation of non-canonical NF-κB signaling pathways.
  • Enhancement of survival and proliferation of mesenchymal stem cells.
  • Mediation of cellular adhesion and metalloproteinase production by synoviocytes in rheumatoid arthritis.
  • Induction of inflammatory responses in endothelial cells and monocytes.

• Cell Culture:
  LIGHT is used to maintain or differentiate stem and immune cells in culture, including promoting the differentiation of human bone marrow-derived mesenchymal stem cells into hepatocyte-like cells.

• Surface Plasmon Resonance (SPR):
  SPR has been used to characterize the binding interactions of LIGHT with its receptors, such as lymphotoxin beta receptor (LTβR) and the decoy receptor DcR3/TR6.

• Tumor Immunology Models:
  Recombinant LIGHT has been applied in mouse tumor models to study anti-tumor immunity, immune cell recruitment, and the effects on the tumor microenvironment. It has been shown to recruit dendritic cells and cytotoxic cells, enhance antigen presentation, and promote effector T cell responses.

• Cell Adhesion Assays:
  LIGHT has been validated for use in cell adhesion assays, particularly in the context of immune cell interactions and inflammatory disease models.

• Biochemical and Biophysical Characterization:
  SDS-PAGE, mass spectrometry, and HPLC have been used to confirm the purity and activity of recombinant LIGHT preparations.

Sample Types Used:

• Whole cells (primary human cells, cell lines)
• Transfected cells
• Whole tissue (e.g., human islets)
• Cell supernatants

Disease and Functional Contexts:

• Rheumatoid arthritis (mediating inflammation and bone resorption)
• Coronary artery disease (regulating inflammatory response)
• Skin fibrosis
• Tumor biology and immunotherapy
• Stem cell biology and differentiation

Summary Table of Validated Applications

Key Insights:

• The most common and well-validated application is bioassay, where LIGHT’s effects on cell signaling, proliferation, apoptosis, and inflammation are measured.
• SPR and cell adhesion assays are used for mechanistic studies of LIGHT-receptor interactions and immune cell function.
• LIGHT is a valuable tool in tumor immunology for studying immune cell recruitment and anti-tumor responses.
• Applications span both basic research (mechanistic studies) and disease models (e.g., rheumatoid arthritis, cancer, cardiovascular disease).

If you require protocols or technical details for any specific application, please specify the experimental context.

To reconstitute and prepare Recombinant Human LIGHT (TNFSF14) protein for cell culture experiments, dissolve the lyophilized protein at a concentration of 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA) if the formulation includes BSA as a carrier, or in sterile PBS alone if the formulation contains trehalose. Avoid vigorous mixing and repeated freeze-thaw cycles to preserve protein activity.

Step-by-step protocol:

• Preparation:

  • Allow both the vial and reconstitution buffer to reach room temperature before opening.
  • Briefly centrifuge or tap the vial to collect all lyophilized material at the bottom.

• Reconstitution:

  • Add sterile PBS (with 0.1% BSA if required) to achieve a final concentration of 100 μg/mL.
  • Gently pipette the solution along the inner wall of the vial to avoid bubbles. Do not vortex; gentle mixing is sufficient.
  • Let the vial sit at room temperature for 15–30 minutes with gentle agitation until the protein is fully dissolved.

• Aliquoting and Storage:

  • Once dissolved, aliquot the solution into smaller volumes to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C to –70°C for long-term storage, or at 2–8°C for short-term use (up to 1 week).
  • For long-term storage at low concentrations, consider adding 5–50% glycerol or using trehalose as a stabilizer.

• Dilution for Cell Culture:

  • Dilute the stock solution to the desired working concentration using cell culture medium or buffer containing carrier proteins (e.g., 0.1% BSA, 5% HSA, or 10% FBS) to minimize adsorption and maintain stability.
  • Do not use water for dilution, as this may cause protein degradation.

Additional notes:

• The effective concentration for cell stimulation (e.g., HUVEC proliferation) is typically in the range of 1–4 ng/mL, but optimal dosing should be empirically determined for your specific assay.
• Always consult the product datasheet or certificate of analysis for formulation-specific instructions, as buffer requirements may vary.

Summary Table: Reconstitution Conditions

Following these steps will ensure optimal solubility and biological activity of recombinant LIGHT protein for cell culture experiments.