Recombinant Rat Fractalkine (Chemokine Domain, aa 25-100)

Recombinant Rat Fractalkine (Chemokine Domain, aa 25-100) is widely used in research applications to study immune cell migration, neuroinflammation, and cell signaling due to its specific interaction with the CX3CR1 receptor and its well-characterized chemotactic and modulatory properties.

Key scientific reasons to use this protein:

• Chemotaxis and Cell Adhesion: The chemokine domain (aa 25-100) of fractalkine is responsible for mediating the chemotactic recruitment and firm adhesion of T cells, monocytes, and NK cells to activated endothelium, making it essential for in vitro and in vivo studies of immune cell trafficking and inflammation.

• Modeling Inflammatory Diseases: Fractalkine plays a proinflammatory role in conditions such as rheumatoid arthritis, ischemia-reperfusion injury, and transplant rejection by inducing synovial angiogenesis, activating monocytes and T cells, and stimulating matrix metalloproteinase production. Its use allows for mechanistic studies and therapeutic target validation in these disease models.

• Neurobiology Research: In the central nervous system, fractalkine is predominantly expressed by neurons and regulates microglial activation via CX3CR1. Recombinant fractalkine has been shown to modulate neurogenesis, suppress excessive microglial activation, and promote hematoma resolution by increasing M2 microglia, making it valuable for neuroinflammation and neurodegeneration studies.

• Biomarker and Prognostic Studies: Fractalkine is an essential biomarker for predicting prognosis in diseases such as colorectal cancer, and its recombinant form is used in assays to quantify and characterize its biological activity.

• Defined Chemokine Domain: Using the chemokine domain (aa 25-100) ensures that the protein retains its receptor-binding and chemotactic functions, while excluding the transmembrane and mucin-like stalk regions, which are not required for most in vitro functional assays.

• High Purity and Activity: Recombinant preparations of this domain are typically highly purified and biologically active, with low endotoxin levels, making them suitable for sensitive cell-based assays and animal studies.

Typical applications include:

• Chemotaxis assays for immune cell migration.
• Cell adhesion studies.
• Investigation of fractalkine-CX3CR1 signaling in neurobiology and immunology.
• Functional studies in models of inflammation, neurodegeneration, and transplantation.
• Use as a standard or control in ELISA and other immunoassays.

In summary, recombinant rat fractalkine (chemokine domain, aa 25-100) is a critical tool for dissecting the molecular mechanisms of immune cell recruitment, neuroimmune interactions, and inflammatory disease processes, with broad utility in both basic and translational research.

You can use recombinant rat Fractalkine (chemokine domain, aa 25-100) as a standard for quantification or calibration in ELISA assays, provided that the ELISA is designed to recognize this domain and the standard is properly validated for your specific assay conditions.

Key considerations:

• Domain specificity: Most commercial ELISA kits for rat Fractalkine (CX3CL1) are designed to detect both natural and recombinant forms, often using the chemokine domain (aa 25-100 or similar) as the standard. If your ELISA uses antibodies that specifically recognize epitopes within this domain, your recombinant protein is suitable as a standard.

• Standard curve generation: The recombinant protein should be reconstituted and diluted according to best practices (e.g., in PBS with carrier protein such as BSA to prevent adsorption), and used to generate a standard curve in the same matrix as your samples to ensure accurate quantification.

• Validation: Confirm that the recombinant standard behaves similarly to endogenous Fractalkine in your assay. This includes parallelism (the standard curve and sample dilution curves should be parallel) and recovery experiments to ensure matrix effects do not interfere with quantification.

• Protein integrity: Ensure the recombinant protein is of high purity, low endotoxin, and properly stored to maintain activity and structure.

• Documentation: Some ELISA kits specify the use of a particular recombinant standard (e.g., aa 22-100 or aa 25-100), and provide protocols for its use. If you are developing your own assay, you can calibrate using your recombinant protein, but you must define the standard curve and validate assay performance parameters (sensitivity, specificity, linearity, etc.).

Summary Table: Use of Recombinant Fractalkine (aa 25-100) as ELISA Standard

Best practices:

• Always validate the recombinant standard in your specific ELISA setup.
• Use the same buffer/matrix for standards and samples to minimize matrix effects.
• Document all standard preparation and validation steps for reproducibility.

If your ELISA kit or protocol specifically requires a different fragment or full-length protein, or if the antibodies used do not recognize the chemokine domain, the recombinant aa 25-100 may not be suitable. Always consult the assay documentation and perform appropriate controls.

Recombinant Rat Fractalkine (Chemokine Domain, aa 25-100) has been validated for several key applications in published research, primarily focusing on its biological activity and utility in immunological and neuroinflammatory studies. The main applications include:

1. Chemotaxis Assays:

   • The protein has been used to chemoattract freshly isolated human peripheral blood lymphocytes (PBL) and BaF3 mouse pro-B cells transfected with human CX3CR1, demonstrating its ability to induce cell migration in a dose-dependent manner.
   • Reference: Leinco product details and R&D Systems validation data.

2. Neutralization Studies:

   • The chemotactic activity of recombinant rat fractalkine can be neutralized by specific antibodies, such as Goat Anti-Rat CX3CL1/Fractalkine Chemokine Domain Antibody (AF537), confirming its functional relevance in experimental settings.
   • Reference: R&D Systems and Bio-Techne antibody product documentation.

3. Microglial Activation Studies:

   • Recombinant rat fractalkine (chemokine domain) has been applied to primary rat microglial cells to study its effects on CX3CR1-mediated signaling, including modulation of pro-inflammatory gene expression (e.g., TNFα, IL-6).
   • Reference: PMC9772123 (CONCENTRATION AND PROTEOLYSIS OF CX3CL1 MAY ...).

4. Western Blot and Immunoassay:

   • The protein is used as a standard or positive control in Western blot and sandwich immunoassay applications to detect and quantify fractalkine expression.
   • Reference: R&D Systems and Bio-Techne antibody product documentation.

5. In Vivo and In Vitro Functional Studies:

   • The chemokine domain of rat fractalkine has been employed in both in vivo and in vitro models to investigate its role in neuroinflammation, synaptic strength, and macrophage survival.
   • Reference: PLoS ONE, Journal of Clinical Investigation, and Proceedings of the National Academy of Sciences.

These applications highlight the utility of recombinant rat fractalkine (chemokine domain, aa 25-100) in studying chemokine biology, immune cell migration, neuroinflammatory processes, and as a tool for functional and neutralization assays.

To reconstitute and prepare Recombinant Rat Fractalkine (Chemokine Domain, aa 25-100) for cell culture experiments, dissolve the lyophilized protein in sterile PBS (phosphate-buffered saline) at a concentration of 100 μg/mL. This concentration is commonly used for stock solutions and allows for accurate dilution to working concentrations for cell assays.

Step-by-step protocol:

• Add sterile PBS directly to the lyophilized protein to achieve a final concentration of 100 μg/mL.
• Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.
• If the protein is difficult to dissolve, allow it to sit at room temperature for a few minutes, then gently mix again.
• After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can reduce protein activity.
• Store aliquots at 4°C for up to 1 week or at –20°C for up to 2 months. For longer-term storage, keep the lyophilized protein at –80°C.
• For cell culture experiments, dilute the stock solution in your cell culture medium to the desired working concentration. The effective concentration for biological activity (e.g., chemotaxis assays) is typically in the range of 3–9 ng/mL (0.003–0.009 μg/mL), but optimal concentrations should be determined empirically for your specific assay.

Additional considerations:

• If your protein preparation does not contain a carrier protein (e.g., BSA), and you observe precipitation or loss of activity, you may add 0.1% BSA to the PBS during reconstitution to stabilize the protein.
• Always use sterile technique to prevent contamination.
• Avoid repeated freeze-thaw cycles by aliquoting the stock solution immediately after reconstitution.

Summary Table

This protocol ensures optimal solubility, stability, and biological activity of recombinant rat Fractalkine for cell culture experiments.