Recombinant Human Fas

Using Recombinant Human Fas in research applications is essential for studying apoptosis, immune regulation, and cell signaling due to its defined activity, high purity, and reproducibility compared to native or non-recombinant sources.

Key reasons to use Recombinant Human Fas:

• Mechanistic Studies of Apoptosis: Fas (also known as CD95 or TNFRSF6) is a critical death receptor that, upon binding its ligand (FasL), triggers the extrinsic apoptotic pathway. Recombinant Human Fas allows precise investigation of Fas-mediated apoptosis in various cell types, including cancer, immune, and primary cells.
• Defined Biological Activity: Recombinant Fas proteins are produced with controlled sequence and post-translational modifications, ensuring consistent and predictable activity in bioassays, such as inducing or inhibiting apoptosis in cell lines like Jurkat cells.
• Reproducibility and Batch Consistency: Recombinant production ensures minimal lot-to-lot variability, which is critical for reproducible results in quantitative assays, mechanistic studies, and drug screening.
• Versatility in Experimental Design: Recombinant Fas can be engineered with tags or fusion partners (e.g., Fc, His) to facilitate detection, purification, or functional studies, and can be used in formats suitable for ELISA, Western blot, flow cytometry, and cell-based assays.
• Ethical and Practical Advantages: Recombinant proteins reduce reliance on animal-derived materials, supporting ethical research practices and simplifying regulatory compliance.
• Cost and Scalability: Recombinant production allows for scalable, cost-effective generation of large quantities of protein, which is often more practical than purifying native Fas from tissues or cell lines.

Common research applications include:

• Inducing or blocking apoptosis in cell culture models to study cell death pathways and immune cell regulation.
• Mapping signaling pathways involving Fas and its downstream effectors.
• Screening for small molecules or biologics that modulate Fas signaling for therapeutic development.
• Using as a standard or control in immunoassays (e.g., ELISA, Western blot).

Technical considerations:

• Recombinant Fas is available in various forms (e.g., soluble, Fc-fusion, tagged), and the choice depends on the intended assay and detection method.
• Biological activity should be validated in relevant cell-based assays, as activity can vary depending on the presence of cross-linkers or the specific cell type used.

In summary, Recombinant Human Fas is a reliable, reproducible, and versatile tool for dissecting Fas-mediated signaling and apoptosis, supporting both basic research and translational applications.

You can use recombinant human Fas as a standard for quantification or calibration in ELISA assays, provided it is immunologically equivalent to the endogenous protein and validated for this purpose. Most commercial ELISA kits for human Fas (CD95/TNFRSF6) use recombinant human Fas as their standard, and these kits are designed to quantify both endogenous and recombinant Fas in biological samples.

Key considerations:

• Immunological Equivalence: The recombinant Fas must be recognized by the antibodies used in your ELISA. Commercial kits confirm that their standards (recombinant Fas) are detected equivalently to endogenous Fas, ensuring accurate quantification.
• Validation: It is essential to validate that your recombinant Fas standard produces a parallel standard curve to endogenous Fas in your assay system. This ensures that quantification is accurate across sample types.
• Recovery and Linearity: Published kit data show high recovery rates (typically 83–101%) for recombinant Fas spiked into serum, plasma, and cell culture supernatants, indicating suitability for calibration.
• Dilution Accuracy: When preparing standards from recombinant protein, large dilution steps (from µg/mL to pg/mL) can introduce error. It is recommended to assign the concentration of your standard based on its measured value in the ELISA, rather than relying solely on the mass stated on the vial.
• Carrier Proteins: Some recombinant Fas preparations contain carrier proteins (e.g., BSA) to stabilize the protein, which may affect assay performance. Carrier-free preparations are preferred for standard curve generation in ELISA.

Best Practices:

• Prepare a standard curve using serial dilutions of recombinant Fas in the same buffer or matrix as your samples.
• Validate the standard curve by spiking known amounts of recombinant Fas into representative sample matrices and assessing recovery and parallelism.
• Use the same recombinant Fas preparation for calibration throughout your experiments to maintain consistency.

Limitations:

• Lot-to-lot variability in recombinant protein preparations may affect quantification; always validate new lots.
• The recombinant Fas standard should be assigned a value based on its performance in your specific ELISA, not just the label concentration.

Summary Table: Recombinant Human Fas as ELISA Standard

In conclusion, recombinant human Fas is suitable as a standard for ELISA quantification, provided you validate its performance in your specific assay system and follow best practices for standard curve preparation and assignment.

Recombinant human Fas has been validated for several key applications in published research:

Functional and Bioassay Applications

Recombinant human Fas proteins have been extensively used in bioassays to measure biological activity. The primary functional assay involves measuring the protein's ability to inhibit Fas ligand-induced apoptosis in cell culture systems, particularly using Jurkat human acute T cell leukemia cells as a standard model. This assay typically measures inhibitory potency with ED₅₀ values in the range of 0.01–0.04 µg/mL in the presence of recombinant human Fas ligand.

Immunological and Diagnostic Applications

ELISA (enzyme-linked immunosorbent assay) represents another validated application, where recombinant soluble Fas can serve dual purposes: as both an active reagent in standard ELISA protocols and as a calibration standard in Fas quantification assays. This application is particularly useful for measuring endogenous Fas levels in biological samples.

Cancer Immunotherapy Research

Recombinant Fas has been validated in cancer immunotherapy applications, particularly in the development of engineered cell therapies. Secreted Fas decoys—derived from the extracellular domain of human Fas—have been engineered into T cells to enhance antitumor activity by sequestering Fas ligand and preventing apoptosis of effector cells in solid tumors. This approach has demonstrated enhanced efficacy in pancreatic cancer models when combined with CAR-T cell technology.

Protein Characterization Applications

Recombinant human Fas has been validated for use in Western blotting (WB) and SDS-PAGE applications, enabling protein detection and characterization studies. Additionally, the protein has been used in blocking assays to investigate the functional role of Fas signaling in various cellular contexts.

Research on Post-Translational Modifications

Recombinant Fas has been employed in studies examining N-glycosylation and its impact on Fas-mediated apoptosis, using advanced analytical techniques such as MALDI-ToF mass spectrometry to characterize glycomic changes.

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

1. Centrifuge the vial
   Before opening, briefly centrifuge the lyophilized protein vial (10,000 × g for 20–30 seconds) to collect all material at the bottom and avoid loss.

2. Reconstitution buffer

   • For most applications, reconstitute the protein at 100 μg/mL in sterile PBS (phosphate-buffered saline).
   • If the formulation contains BSA (bovine serum albumin) as a carrier, use PBS containing at least 0.1% BSA or human serum albumin to stabilize the protein and prevent adsorption to plastic surfaces.
   • If the protein is carrier-free, sterile PBS alone is sufficient.

3. Dissolving the protein

   • Gently add the appropriate volume of buffer directly to the vial.
   • Allow the vial to sit at room temperature for 10–15 minutes.
   • Gently swirl or invert the vial to mix. Do not vortex, as this can denature the protein.

4. Aliquoting and storage

   • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
   • Store aliquots at –20°C or below for long-term storage. For short-term use (up to one week), store at 2–8°C.
   • If storing for longer periods, ensure the presence of carrier protein (e.g., BSA) to maintain stability.

5. Preparation for cell culture

   • Before adding to cell cultures, dilute the reconstituted protein to the desired working concentration using cell culture medium.
   • For functional assays, typical working concentrations range from 0.01–0.04 μg/mL in the presence of recombinant human Fas Ligand, but optimal concentrations should be determined empirically for your specific assay.

Additional notes:

• Always use sterile technique throughout the process to prevent contamination.
• If the protein is to be used in sensitive assays or in vivo, confirm that all buffers and reagents are endotoxin-free.
• Avoid repeated freeze-thaw cycles by aliquoting into single-use volumes.

Summary Table: Recombinant Human Fas Protein Reconstitution

These guidelines are based on standard protocols for recombinant Fas protein and should be adapted as needed for your specific experimental requirements.