Recombinant Human TNF-β

Recombinant Human TNF-β (also known as lymphotoxin-alpha, LT-α) is used in research applications to study its roles in inflammation, immune regulation, lymphoid organ development, and cytotoxicity against tumor cells.

Key reasons to use Recombinant Human TNF-β in research:

• Modeling Inflammatory and Immune Responses: TNF-β is a potent mediator of inflammatory and immune responses, acting through TNF receptors (TNFR1 and TNFR2) to regulate immune cell activation, cytokine production, and cell death pathways.
• Lymphoid Organogenesis: TNF-β is essential for the development and organization of secondary lymphoid organs, such as lymph nodes and Peyer’s patches, making it valuable for developmental immunology studies.
• Tumor Cytotoxicity: Like TNF-α, TNF-β can induce cytotoxic effects on certain tumor cells, supporting research into cancer biology and potential anti-tumor therapies.
• Autoimmunity and Demyelination Models: TNF-β is implicated in autoimmune disorders and inflammatory demyelination, making it relevant for studying diseases such as multiple sclerosis and rheumatoid arthritis.
• Bioassays and Functional Studies: Recombinant TNF-β is used in cell-based assays to assess cytokine signaling, cytotoxicity, and immune cell function, providing a controlled and reproducible reagent for mechanistic studies.

Additional considerations:

• Recombinant proteins offer high purity, batch-to-batch consistency, and defined activity, which are critical for reproducible experimental results.
• Using human recombinant TNF-β ensures species specificity, which is important for translational research and human cell-based assays.

In summary, Recombinant Human TNF-β is a versatile tool for dissecting the molecular and cellular mechanisms of inflammation, immunity, lymphoid tissue development, and tumor biology in both basic and translational research contexts.

Recombinant Human TNF-β can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system.

Most commercial ELISA kits for human TNF-β (also known as lymphotoxin-alpha) are designed to recognize both natural and recombinant forms of the protein, and they typically use recombinant TNF-β as the calibration standard for generating the standard curve. This allows for accurate quantification of TNF-β in biological samples by comparing sample signals to those generated by known concentrations of the recombinant standard.

Key considerations for using recombinant TNF-β as an ELISA standard:

• Parallelism: The assay should demonstrate parallel dose-response curves between the recombinant standard and endogenous TNF-β in your sample matrix. This ensures that the antibodies in the kit recognize both forms equivalently, allowing for accurate quantification.
• Standard Preparation: Prepare the recombinant TNF-β standard according to the manufacturer’s instructions, typically by serial dilution in the same buffer used for your samples to minimize matrix effects.
• Validation: If you are developing your own ELISA or modifying a commercial kit, validate that your recombinant TNF-β standard produces a linear and reproducible standard curve within the assay’s dynamic range.
• Formulation: Recombinant TNF-β is often supplied lyophilized and may contain carrier proteins such as BSA to enhance stability. Reconstitute and store the standard as recommended to maintain activity and consistency.

Best practices:

• Always run a fresh standard curve with each assay batch to account for potential variability in protein stability or assay conditions.
• Confirm that your recombinant TNF-β is of high purity and correctly folded, as misfolded or degraded protein may not be recognized by the ELISA antibodies.
• If available, correlate your standard to an international reference standard (e.g., NIBSC/WHO) for greater comparability across studies.

In summary, recombinant human TNF-β is widely accepted and routinely used as a standard for ELISA quantification, provided the assay is validated for parallel recognition of recombinant and natural protein forms.

Recombinant Human TNF-β (Lymphotoxin-α) has been validated for a range of applications in published research, primarily in bioassays, cell-based functional studies, and various immunological and inflammation-related assays.

Key validated applications include:

• Bioassays for cytotoxicity: TNF-β induces cytotoxicity in cell lines such as L-929 mouse fibroblasts, especially in the presence of actinomycin D, serving as a standard assay for TNF-β activity.
• Cell signaling and receptor activation studies: TNF-β homotrimers activate TNF receptors (TNFRSF1A, TNFRSF1B, HVEM/TNFRSF14), and heterotrimers with LT-β activate LT-βR/TNFRSF3, making it useful for dissecting receptor-mediated signaling pathways.
• Immunological research: TNF-β is used to study lymph node development, inflammation, autoimmune disease mechanisms, and immune cell regulation, including dendritic cell and NK cell interactions.
• Inflammation and autoimmune disease models: TNF-β has been applied in models of inflammatory demyelination, rheumatoid arthritis, lupus, and other autoimmune conditions.
• Cancer research: TNF-β is used to investigate its role in cell proliferation, apoptosis, and tumor microenvironment modulation.
• Leukocyte transmigration and vascular biology: TNF-β has been used to study mechanisms of leukocyte movement across endothelial barriers in inflammation.
• Antibody and inhibitor validation: Recombinant TNF-β is employed in binding and neutralization assays to validate therapeutic antibodies and nanobodies targeting TNF pathways.
• Protein detection and quantification: TNF-β is used as a standard in ELISA, Western blot, immunoprecipitation, flow cytometry, and immunofluorescence protocols for protein quantification and localization.

Summary Table of Validated Applications

Additional Notes:

• TNF-β is frequently used in whole cell assays and in vitro systems, but also in animal models for mechanistic studies.
• Its role in mediating inflammatory and immune responses makes it a standard reagent for validating new therapeutics and studying disease mechanisms.

If you require protocol details or specific assay conditions for any application, please specify the intended use.

To reconstitute and prepare Recombinant Human TNF-β (Lymphotoxin-alpha) 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). This carrier protein helps stabilize TNF-β and prevents adsorption to plasticware.

Step-by-step protocol:

• Equilibrate the vial and reconstitution buffer (PBS + 0.1% BSA) to room temperature before opening.
• Centrifuge the vial briefly to collect the lyophilized powder at the bottom.
• Add buffer: For example, to reconstitute 10 μg of protein, add 100 μL of sterile PBS with 0.1% BSA to achieve 100 μg/mL.
• Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.
• Incubate at room temperature for 15–30 minutes to ensure complete dissolution. If visible flakes remain, continue gentle mixing for up to 2 hours.
• Aliquot the reconstituted solution into working volumes to avoid repeated freeze-thaw cycles.
• Storage: Store aliquots at ≤ –20°C for long-term use. For short-term use (up to 1 week), store at 2–8°C. Avoid repeated freeze-thaw cycles.

Preparation for cell culture:

• Dilute the stock solution to the desired working concentration using cell culture medium containing a carrier protein (e.g., 0.1% BSA or heat-inactivated FCS) to minimize adsorption and loss.
• Determine optimal concentration for your specific assay by performing a dose-response experiment, as effective concentrations may vary (e.g., ED50 for TNF-β bioactivity is typically 0.1–0.4 ng/mL).
• Sterility: Ensure all solutions and containers are sterile to prevent contamination.

Additional notes:

• If the protein is supplied carrier-free, reconstitute in sterile distilled water to 0.1–1.0 mg/mL, then dilute in buffered solution with carrier protein for cell culture.
• Always consult the product-specific Certificate of Analysis (CoA) or datasheet for any unique instructions.
• Avoid sodium azide in functional assays, as it can be cytotoxic.

Summary Table:

This protocol ensures maximum stability and bioactivity of recombinant TNF-β for cell culture applications.