Recombinant Human TWEAK R

Recombinant Human TWEAK R (also known as Fn14 or TNFRSF12A) is a valuable tool for research applications due to its critical role in mediating the biological effects of TWEAK (TNFSF12), a member of the TNF superfamily. Here are several reasons why you should consider using Recombinant Human TWEAK R in your research:

1. Study TWEAK/Fn14 Signaling Pathways

Recombinant TWEAK R allows you to investigate the molecular mechanisms underlying TWEAK-induced signaling, including NF-κB activation, cell proliferation, migration, survival, and apoptosis. This is essential for understanding how TWEAK/Fn14 axis regulates cellular responses in various physiological and pathological contexts.

2. Modulate TWEAK Activity

TWEAK R can be used as a decoy receptor to inhibit TWEAK-dependent effects. For example, recombinant TWEAK R Fc chimera proteins have been shown to block TWEAK-induced proliferation of endothelial cells, making them useful for studying the functional consequences of TWEAK neutralization in vitro and in vivo.

3. Investigate Disease Mechanisms

The TWEAK/Fn14 axis has been implicated in several diseases, including:

• Autoimmune and inflammatory diseases (e.g., lupus, multiple sclerosis)
• Cancer (e.g., melanoma, renal cell carcinoma, breast cancer)
• Cardiovascular diseases (e.g., ischemia/reperfusion injury, heart failure)
• Neurodegenerative disorders

Using recombinant TWEAK R enables researchers to dissect the contribution of this pathway to disease progression and to evaluate potential therapeutic strategies targeting Fn14.

4. Develop and Test Therapeutics

Recombinant TWEAK R can be used in preclinical studies to assess the efficacy of anti-TWEAK or anti-Fn14 therapies. For instance, it can serve as a positive control in assays designed to screen for inhibitors of TWEAK/Fn14 interaction or to validate the specificity of neutralizing antibodies.

5. Support 3D Cell and Organoid Models

In advanced cell culture systems such as organoids, recombinant TWEAK R can help modulate the tumor microenvironment or inflammatory responses, providing more physiologically relevant models for drug screening and disease modeling.

6. High Purity and Consistency

Recombinant proteins are produced under controlled conditions, ensuring batch-to-batch consistency, high purity, and defined activity. This reliability is crucial for reproducible experimental results and regulatory compliance in translational research.

7. Versatile Applications

Recombinant TWEAK R can be used in a variety of assays, including:

• Binding studies (e.g., surface plasmon resonance, ELISA)
• Functional assays (e.g., inhibition of TWEAK-induced proliferation or cytokine secretion)
• Structural studies (e.g., crystallography, NMR)

In summary, Recombinant Human TWEAK R is an indispensable reagent for researchers studying the TWEAK/Fn14 pathway, its role in health and disease, and the development of targeted therapeutics. Its ability to modulate TWEAK activity makes it a powerful tool for both basic and applied research.

Yes, recombinant Human TWEAK R can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for cytokines and receptors.

Essential context and best practices:

• Standard Curve Requirement: For quantitative ELISA, a standard curve must be generated using known concentrations of the analyte. Recombinant proteins are routinely used for this purpose, especially when purified native protein is unavailable.
• Purity and Quantification: The recombinant Human TWEAK R should be highly purified. Its concentration should be verified, ideally by an orthogonal method such as HPLC or UV absorbance, to ensure accuracy in calibration.
• Formulation Considerations: Recombinant proteins formulated with stabilizers (e.g., BSA) are often recommended for use as ELISA standards to improve stability and reproducibility.
• Validation: It is important to validate the standard curve generated with the recombinant protein in your specific ELISA system. This includes confirming linearity, sensitivity, and recovery within the assay’s dynamic range.
• Kit Compatibility: If using a commercial ELISA kit, ensure that the recombinant Human TWEAK R matches the analyte detected by the kit (e.g., same isoform, tag, or domain). Some kits provide their own recombinant standard, which is optimized for the assay.

Additional relevant information:

• Potential Discrepancies: Be aware that mass values calculated by ELISA may differ from those reported on the recombinant protein vial due to dilution errors or differences in protein activity.
• Documentation: Always document the source, lot, and concentration determination method for your recombinant standard to ensure reproducibility and traceability.

In summary, recombinant Human TWEAK R is suitable as an ELISA standard if it is pure, accurately quantified, and validated within your assay system.

Recombinant Human TWEAK R (also known as TWEAK receptor, Fn14, or TNFRSF12A) has been validated in published research for several key applications, primarily in the context of studying TWEAK–TWEAKR signaling and its biological effects.

Validated Applications in Published Research:

• Bioassays / Functional Assays:
  Used to study cell proliferation, migration, apoptosis, and cytokine induction in various cell types, especially endothelial cells and keratinocytes. These assays often assess the biological activity of TWEAK–TWEAKR interactions, such as promoting endothelial cell growth, migration, and angiogenesis, or mediating inflammatory responses and cell death.

• Surface Plasmon Resonance (SPR):
  Employed to characterize protein–protein interactions, specifically the binding affinity between TWEAK and its receptor, or to screen for inhibitors of this interaction.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Used as a standard or calibrator in immunoassays to quantify TWEAK or TWEAKR in biological samples, and to validate antibody specificity.

• Western Blot:
  Applied for protein detection and quantification in cell lysates or tissue samples, confirming the presence and size of TWEAKR.

• Blocking Assays:
  Utilized to inhibit TWEAK-mediated signaling in functional studies, helping to dissect the role of TWEAKR in various cellular processes.

• qPCR (Quantitative PCR):
  Used to measure gene expression changes in response to TWEAKR stimulation, particularly in studies of inflammatory gene induction in human cells.

Representative Research Contexts:

• Endothelial Cell Biology:
  TWEAKR is used to study endothelial cell proliferation, migration, and angiogenesis.

• Inflammation and Autoimmunity:
  Functional assays with TWEAKR have elucidated its role in inflammatory signaling, cytokine secretion, and autoimmune disease models (e.g., psoriasis, lupus, atopic dermatitis).

• Cancer Research:
  TWEAKR is investigated for its involvement in tumor cell invasion, cancer stem cell properties, and as a therapeutic target in melanoma and liver cancer.

• Protein–Protein Interaction Studies:
  SPR and ELISA formats are used to quantify and characterize TWEAK–TWEAKR binding and to screen for potential inhibitors.

Summary Table of Validated Applications

Additional Notes:

• TWEAKR is frequently used in studies involving whole cells (e.g., keratinocytes, endothelial cells) and recombinant protein formats.
• It is a critical tool for dissecting the TWEAK–TWEAKR axis in inflammation, tissue regeneration, and cancer biology.

If you need protocols or more detailed application notes for a specific assay, please specify the context or experimental system.

To reconstitute and prepare Recombinant Human TWEAK R protein for cell culture experiments, follow these steps to ensure protein integrity and biological activity:

1. Storage and Preparation

   • Store the lyophilized protein at -20°C to -80°C until use.
   • Before opening, briefly centrifuge the vial to collect all powder at the bottom.

2. Reconstitution

   • Use the buffer recommended in the product datasheet or Certificate of Analysis (COA). Commonly, sterile PBS or sterile distilled water is used, but always confirm with the specific documentation for your protein.
   • Typical reconstitution concentrations are 0.1–1.0 mg/mL. For example, add 100–1000 µL buffer to 100 µg protein to achieve 1–0.1 mg/mL.
   • Gently add the buffer along the vial wall to avoid foaming and bubbles.
   • Do not vortex or shake vigorously; gently pipette or swirl to dissolve.
   • Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation. If particulates remain, mix at room temperature for up to 2–3 hours or overnight at 4°C.

3. Aliquoting and Storage

   • Once fully dissolved, aliquot the solution into small volumes (≥20 µL) to avoid repeated freeze-thaw cycles.
   • For short-term use (up to one week), store at 2–8°C.
   • For long-term storage, dilute with a buffer containing a carrier protein (e.g., 0.1% BSA, 5% HSA, or 10% FBS) to stabilize the protein, then store aliquots at -20°C to -80°C.

4. Dilution for Cell Culture

   • Before adding to cell culture, further dilute the reconstituted protein to the desired working concentration using culture medium or buffer containing carrier protein.
   • Avoid using pure water for dilution, as this can cause protein degradation.

5. Special Considerations

   • For serum-free cultures or in vivo experiments, avoid animal-derived carriers; use alternatives like trehalose for stabilization.
   • Always consult the product datasheet or COA for specific instructions regarding buffer composition, concentration, and storage conditions.

Summary of Best Practices:

• Centrifuge vial before opening.
• Reconstitute in recommended buffer (often PBS or sterile water).
• Gently mix, avoid vortexing.
• Aliquot and store appropriately.
• Use carrier proteins for dilution and long-term storage.
• Follow datasheet/COA for specific instructions.

These steps will help maintain the biological activity and stability of Recombinant Human TWEAK R protein for reliable cell culture experiments.