Recombinant Mouse Fas

Recombinant Mouse Fas (CD95/TNFRSF6) is used in research applications to study and manipulate apoptosis, immune regulation, and cell signaling with high specificity and reproducibility. Its recombinant form ensures consistent activity and purity, which is critical for experimental reliability.

Key scientific reasons to use Recombinant Mouse Fas:

• Apoptosis Studies: Fas is a death receptor that triggers programmed cell death upon binding to Fas Ligand (FasL). Recombinant Mouse Fas enables controlled induction or inhibition of apoptosis in Fas-expressing cells, allowing researchers to dissect cell death pathways, immune cell clearance, and mechanisms of tissue homeostasis.
• Immune Regulation: Fas-mediated apoptosis is essential for eliminating virus-infected or autoreactive cells, contributing to immune tolerance and shutdown of immune responses. Recombinant Fas can be used to model these processes in vitro or in vivo, aiding studies of autoimmunity, infection, and cancer immunology.
• Functional Assays: Recombinant Mouse Fas is commonly used in assays to measure FasL-induced apoptosis, screen for Fas/FasL pathway modulators, and validate antibody specificity. Its defined structure and activity allow for quantitative and reproducible results.
• Protein Engineering and Mechanistic Studies: The recombinant format allows for fusion tags (e.g., Fc chimeras) that facilitate purification, detection, and functional studies, such as receptor-ligand binding assays or cell signaling investigations.
• Batch-to-Batch Consistency: Recombinant proteins provide uniformity in experimental conditions, minimizing variability and enhancing reproducibility compared to native or non-recombinant sources.

Typical applications include:

• Inducing apoptosis in Fas-expressing cell lines to study cell death mechanisms.
• Investigating immune cell clearance and tolerance in mouse models.
• Screening for drugs or antibodies that modulate Fas signaling.
• Using as a standard in ELISA or flow cytometry for quantifying Fas or FasL interactions.

Best practices:

• Select the appropriate recombinant Fas format (e.g., Fc chimera, carrier-free) based on your assay requirements.
• Validate activity in your specific cell system, as Fas-induced apoptosis can be rapid and dose-dependent.
• Use recombinant Fas in combination with FasL or pathway inhibitors to dissect signaling mechanisms.

In summary, Recombinant Mouse Fas is a precise tool for apoptosis and immune regulation research, offering high specificity, reproducibility, and flexibility for mechanistic and translational studies.

You can use recombinant Mouse Fas protein as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with your assay’s antibodies and detection system.

Key considerations and best practices:

• Purity and Quantification: The recombinant Mouse Fas should be highly purified, and its concentration must be precisely known, typically determined by absorbance at 280 nm, BCA, or Bradford assay.
• Formulation: If possible, use a carrier-free or BSA-containing formulation depending on your application. Carrier-free is preferred for standard curve preparation to avoid interference, while BSA-containing formulations are often used for cell culture or as blocking agents.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant protein in the same buffer or matrix as your samples to generate a standard curve covering the expected concentration range of your analyte.
• Antibody Compatibility: Ensure that the recombinant Fas protein contains the same epitope(s) recognized by the capture and detection antibodies in your ELISA. Most commercial ELISA kits use recombinant proteins as standards, but the expression system and protein fragment used should match the kit’s specifications.
• Validation: It is good practice to validate the recombinant standard in your specific ELISA format, confirming that it produces a linear, reproducible standard curve and that its signal is comparable to endogenous Fas in your sample matrix.

Summary Table: Recombinant Mouse Fas as ELISA Standard

Additional notes:

• Many commercial ELISA kits for Mouse Fas or Fas Ligand use recombinant proteins as their standards, indicating this is a widely accepted practice.
• Always consult your ELISA kit’s manual for any specific requirements regarding the standard protein’s sequence, expression system, or formulation.

In summary, recombinant Mouse Fas is suitable as a standard for ELISA quantification if it is well-characterized and compatible with your assay system.

Recombinant Mouse Fas (CD95/TNFRSF6) has been validated in published research for applications including apoptosis assays, cell viability/cytotoxicity assays, receptor-ligand binding studies, and as a tool in immunological and cancer research models.

Key validated applications include:

• Apoptosis induction assays: Recombinant Mouse Fas is widely used to trigger apoptosis in Fas-sensitive cell lines, such as Jurkat T cells, to study cell death pathways and screen for apoptosis modulators. The protein’s ability to induce apoptosis is often quantified by measuring cell viability or caspase activation.

• Cell viability/cytotoxicity assays: In engineered reporter cell lines expressing chimeric receptors (e.g., mHVEM:hFas), recombinant Fas or its ligand is used to assess cytotoxic responses and screen for modulators of Fas-mediated cell death.

• Receptor-ligand binding studies: Recombinant Fas (often as an Fc chimera) is used in binding assays to characterize interactions with Fas ligand (FasL) or to block FasL-mediated signaling in vitro.

• Immunological research: Recombinant Mouse Fas is used to study immune regulation, particularly in the context of T cell homeostasis, immune privilege, and mechanisms of immune evasion in cancer or transplantation models.

• Cancer research: Recombinant Fas and its ligand are used to investigate tumor cell sensitivity to Fas-mediated apoptosis, the role of Fas signaling in tumor immune escape, and to test therapeutic strategies involving Fas pathway modulation.

• ELISA and Western blot standards: Recombinant Fas is used as a standard or positive control in immunoassays to quantify Fas or FasL levels in biological samples.

• Flow cytometry and immunoprecipitation: Recombinant Fas can be used as a reagent to detect or pull down FasL or to block Fas-FasL interactions in cell-based assays.

Supporting details from published research:

• In gene therapy studies, recombinant mouse Fas ligand was used to induce apoptosis in target cells and to modulate immune responses in vivo, demonstrating its utility in both mechanistic and therapeutic research.
• Recombinant Fas and FasL proteins have been used in cell-based reporter assays to study cytotoxicity and receptor signaling, including in engineered cell lines expressing chimeric receptors.
• Apoptosis assays using recombinant Fas have been validated in multiple studies, including as a positive control for cell death induction and for screening apoptosis inhibitors.
• Recombinant Fas-Fc chimeras are commonly used in binding and blocking studies to dissect Fas/FasL interactions.

Summary of main validated applications:

If you need details on a specific application or protocol, please specify the context or experimental system.

To reconstitute and prepare Recombinant Mouse Fas protein for cell culture experiments, follow these general guidelines based on manufacturer protocols and best practices for recombinant proteins:

1. Reconstitution

• Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
• Reconstitute the lyophilized protein with sterile, cold PBS (pH 7.2–7.4) or sterile distilled water, unless the product datasheet specifies a different buffer.
  • Typical reconstitution concentration: 0.1–0.5 mg/mL (check datasheet for exact recommendation).
  • Example: For a 10 µg vial, add 20–100 µL of buffer to achieve 0.1–0.5 mg/mL.
• Gently swirl or rotate the vial to dissolve the protein. Avoid vigorous shaking to prevent foaming and denaturation.
• Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation for complete dissolution.

2. Addition of Carrier Protein (Optional but Recommended)

• For increased stability, especially in long-term experiments, add 0.1–1% BSA (Bovine Serum Albumin) to the reconstituted protein solution.
• This helps prevent protein loss due to adsorption to surfaces.

3. Aliquoting and Storage

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –80°C for long-term storage (typically stable for 6–12 months).
• For short-term use (up to 1 week), store at 2–8°C.

4. Preparation for Cell Culture

• Dilute the reconstituted protein in cell culture medium or assay buffer to the desired working concentration.
• Filter-sterilize (0.22 µm filter) if necessary before adding to cell cultures.
• Use immediately after dilution for best activity.

5. Handling Tips

• Avoid repeated freeze-thaw cycles.
• Use low-binding tubes to minimize protein loss.
• Always refer to the specific product datasheet for exact reconstitution instructions, as formulations may vary between suppliers.

Summary:

• Reconstitute with sterile PBS or water (0.1–0.5 mg/mL).
• Add 0.1–1% BSA for stability.
• Aliquot and store at –20°C or –80°C.
• Dilute in culture medium before use.

Always consult the product datasheet for the specific recombinant Fas protein you are using, as some may have unique requirements.