Recombinant Human TWEAK

Recombinant Human TWEAK (TNFSF12) is widely used in research because it is a well-characterized cytokine that enables precise, reproducible studies of cell signaling, inflammation, tissue regeneration, and disease mechanisms, particularly those involving the TWEAK/Fn14 pathway.

Key reasons to use recombinant human TWEAK in research applications:

• Defined Bioactivity and Consistency: Recombinant TWEAK provides a pure, quantifiable, and consistent source of the protein, essential for reproducible results in cell-based assays, bioassays, and biochemical studies.
• Functional Studies: It is used to investigate TWEAK’s roles in cell proliferation, migration, survival, differentiation, and apoptosis across various cell types, including endothelial, epithelial, and immune cells.
• Disease Modeling: TWEAK is implicated in inflammation, autoimmune diseases, cancer, tissue injury, and regeneration. Recombinant TWEAK allows researchers to model these processes in vitro and in vivo, such as studying its effects on liver regeneration, wound healing, or tumor microenvironment dynamics.
• Pathway Analysis: TWEAK’s interaction with its receptor Fn14 activates signaling pathways like NF-κB and STAT3, making it valuable for dissecting molecular mechanisms underlying cytokine signaling and downstream gene expression.
• Assay Development: Recombinant TWEAK is used as a standard in immunoassays (e.g., ELISA) and as a positive control in gene expression or protein quantification assays, ensuring assay sensitivity and specificity.
• Organoid and 3D Culture Systems: Its defined activity supports advanced models such as organoids, where batch-to-batch consistency and high bioactivity are critical for studying tissue development and disease.

Additional considerations:

• Carrier-free and BSA-containing forms are available for different applications, such as cell culture or ELISA standards, to minimize background or optimize protein stability.
• Recombinant TWEAK is not intended for clinical or diagnostic use but is strictly for research purposes.

In summary, using recombinant human TWEAK enables controlled, mechanistic studies of its biological functions and disease relevance, supporting applications in immunology, oncology, regenerative medicine, and assay development.

Yes, you can use recombinant human TWEAK as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common and accepted practice in quantitative ELISA protocols for cytokines and growth factors.

Key considerations and supporting details:

• Recombinant proteins are widely used as ELISA standards: Guidelines for ELISA standard preparation recommend using a purified protein, and recombinant proteins are specifically mentioned as suitable when purified native protein is unavailable. The recombinant TWEAK should be well-characterized, with a known concentration, and ideally supplied or validated for use as an ELISA standard.

• Commercial ELISA kits and protocols use recombinant TWEAK as the calibrator: Multiple ELISA kits for human TWEAK (TNFSF12) use recombinant human TWEAK as the standard for generating the calibration curve. These kits demonstrate that recombinant TWEAK is recognized equivalently to native TWEAK in the assay, allowing for accurate quantification.

• Parallelism and validation: For best results, confirm that the recombinant TWEAK standard shows parallelism with your sample matrix (e.g., serum, plasma, or cell culture supernatant). This ensures that the standard and endogenous TWEAK behave similarly in the assay, which is critical for accurate quantification.

• Preparation and handling: Follow best practices for reconstitution, dilution, and storage of the recombinant standard. Use the same buffer as your samples or the recommended standard diluent to minimize matrix effects.

• Documentation and traceability: Record the lot number, concentration, and source of the recombinant TWEAK used as a standard for reproducibility and troubleshooting.

Summary of best practices:

• Use a highly purified, well-characterized recombinant human TWEAK.
• Validate that the standard curve generated with recombinant TWEAK is linear and covers the expected concentration range of your samples.
• Confirm parallelism between the standard and sample curves, especially if your sample matrix differs from the standard diluent.
• Prepare and store the standard according to manufacturer or protocol recommendations to maintain stability and activity.

In conclusion, recombinant human TWEAK is suitable and commonly used as a standard for ELISA quantification, provided these technical considerations are addressed.

Recombinant Human TWEAK has been validated for a range of applications in published research, primarily in the context of cell signaling, inflammation, and immunology. The most commonly reported applications include:

• Bioassays / Functional Assays: Used to study cell proliferation, apoptosis, cytokine induction, and inflammatory signaling in various human cell types, including keratinocytes, hepatic stellate cells, and cancer cell lines.
• Surface Plasmon Resonance (SPR): Applied to characterize protein-protein interactions, such as binding to its receptor Fn14 or to neutralizing antibodies.
• ELISA (Immunoassay Standard): Used as a calibrator or standard in ELISA assays to quantify TWEAK levels in biological samples like serum and plasma.
• Blocking Assays: Employed to assess the inhibition of TWEAK-mediated signaling, often in the context of therapeutic antibody development.
• Western Blot: Used as a positive control or to confirm protein expression and molecular weight.
• qPCR (TaqMan): Utilized to induce gene expression changes in treated cells, with downstream quantification by qPCR.

In vivo and in vitro research applications include:

• Wound Healing and Angiogenesis: Recombinant TWEAK has been shown to promote angiogenesis and accelerate wound healing in diabetic mouse models, with effects on endothelial cell proliferation, migration, and tubule formation.
• Inflammation Models: Used to induce and study skin inflammation, atopic dermatitis, and psoriasis in mouse models and human keratinocytes, often in synergy with cytokines like TNF and IL-17A.
• Cancer Research: Investigated for its role in tumor cell invasion, cancer stem cell properties, and as a target for immunotoxin therapies.
• Autoimmune and Tissue Injury Models: Applied in studies of systemic lupus erythematosus, cerebral ischemia, and tissue repair, often to modulate or assess TWEAK/Fn14 signaling.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, demonstrating the versatility of recombinant human TWEAK in both basic and translational research.

Reconstitution Protocol

Recombinant Human TWEAK protein is typically supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The reconstitution process is critical for maintaining protein activity and ensuring reliable experimental results.

Initial Preparation

Begin by allowing both the lyophilized vial and reconstitution buffer to equilibrate to room temperature. Perform a brief centrifugation (3000-3500 rpm for 5 minutes) or gently tap the vial to ensure all lyophilized material settles to the bottom. This step prevents loss of protein material during reconstitution.

Reconstitution Steps

Add the appropriate volume of sterile PBS (or sterile distilled water, depending on your specific product formulation) to achieve the recommended concentration, typically 100 μg/mL. Add the buffer solution slowly along the inner wall of the vial to minimize bubble formation. Allow the vial to sit at room temperature for approximately 20 minutes with gentle agitation. This incubation period ensures complete dissolution of the protein. Do not vortex the solution, as vigorous shaking can break chemical bonds and inactivate the protein.

Gently pipette the solution several times or blow gently with a pipette to facilitate complete dissolution. The reconstituted solution should appear clear. If flakes or particulates remain visible, continue mixing for a couple of hours at room temperature, then overnight at 4°C.

Storage and Stability

Short-term Storage

For experiments requiring use within one week, store the reconstituted protein at 4°C under sterile conditions. This storage duration is suitable for standard cell culture experiments with 5-7 day cycles.

Long-term Storage

For extended storage, prepare aliquots of at least 20 μL each to minimize protein loss from adsorption to container walls. Store these aliquots at -20°C to -80°C. Under these conditions, the protein remains stable for 3-6 months. Avoid repeated freeze-thaw cycles, as each cycle decreases protein activity.

Dilution for Cell Culture

After initial reconstitution, further dilutions should be made using culture medium or PBS containing carrier proteins or stabilizers. Appropriate carriers include bovine serum albumin (BSA) at 0.1-5% concentration, fetal bovine serum (FBS) at 5-10%, or human serum albumin (HSA). These carriers prevent protein adsorption and maintain stability at lower concentrations.

For serum-free culture applications or in vivo experiments where animal-derived proteins must be avoided, use trehalose as the stabilizer instead. Trehalose effectively preserves protein activity during storage and can extend stability even at room temperature.

Functional Considerations

Recombinant Human TWEAK is a 17.0 kDa soluble polypeptide comprising 154 amino acid residues representing the TNF-homologous region. The protein demonstrates biological activity with an ED₅₀ of 2-8 ng/mL in stimulating proliferation of human umbilical vein endothelial cells (HUVEC). This potency range should inform your dilution calculations for dose-response experiments.