Recombinant Rat Fractalkine

Recombinant Rat Fractalkine (CX3CL1) is a valuable tool for research applications focused on neuroinflammation, chemokine signaling, immune cell migration, and disease models involving the central nervous system and inflammatory processes. It enables controlled, reproducible studies of fractalkine’s biological functions and receptor interactions.

Key reasons to use recombinant rat fractalkine in research:

• Functional Studies of Chemokine Signaling: Recombinant rat fractalkine specifically binds to and activates the CX3CR1 receptor, allowing detailed investigation of the CX3CL1–CX3CR1 axis in rat models. This is critical for dissecting mechanisms of neuron–microglia communication, immune cell trafficking, and inflammatory responses in the CNS and peripheral tissues.

• Disease Modeling and Therapeutic Evaluation: Administration of recombinant fractalkine in vivo has been shown to modulate neuroinflammation, enhance hematoma resolution, and improve neurological outcomes in rat models of brain injury, such as germinal matrix hemorrhage. This makes it a useful reagent for preclinical studies of neuroprotection, neurodegeneration, and inflammatory diseases.

• Bioactivity and Assay Development: Recombinant rat fractalkine is validated for bioactivity, including chemotaxis assays, receptor binding, and functional cell-based assays. It can be used as a standard in ELISA, Western blot, and immunoassays to quantify endogenous fractalkine or to neutralize its activity in mechanistic studies.

• Reproducibility and Consistency: Using recombinant protein ensures batch-to-batch consistency, defined concentration, and absence of confounding factors present in tissue extracts, which is essential for reproducible experimental results.

• Versatility in Experimental Design: Recombinant fractalkine is available in different forms (e.g., chemokine domain, full-length, carrier-free), suitable for a range of applications including in vitro cell culture, in vivo animal studies, and biochemical assays.

• Immunological and Inflammatory Research: Fractalkine is implicated in leukocyte adhesion, migration, and inflammatory disease pathogenesis, such as rheumatoid arthritis and cardiovascular complications. Recombinant protein allows for direct testing of its role in these processes and for screening potential therapeutic interventions.

In summary, recombinant rat fractalkine is essential for mechanistic, translational, and therapeutic research where precise control over chemokine signaling is required, especially in rat-based models of neurobiology and inflammation.

Yes, recombinant Rat Fractalkine (CX3CL1) protein can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the form detected by your assay.

Supporting details:

• ELISA kits for Rat Fractalkine (CX3CL1) typically use recombinant protein as the standard for generating calibration curves. These kits are designed to recognize both natural and recombinant forms of the protein, ensuring accurate quantification in biological samples.
• The recombinant standard is included in commercial ELISA kits and is used to establish the standard curve, which allows for quantification of Fractalkine in unknown samples.
• Recombinant proteins formulated with carrier proteins (e.g., BSA) are recommended for use as ELISA standards, as they improve stability and reproducibility.
• It is essential to ensure that the recombinant protein you use as a standard is of high purity, properly quantified, and matches the epitope recognized by the antibodies in your ELISA system.
• The standard curve generated with recombinant Fractalkine should be linear and parallel to curves obtained with natural protein, confirming suitability for quantification.

Best practices:

• Reconstitute the recombinant protein according to the manufacturer’s instructions, typically in PBS with carrier protein (e.g., 0.1% BSA), to prevent adsorption and loss.
• Validate that your ELISA antibodies recognize the recombinant form equivalently to the native protein. Most commercial kits are designed for this, but custom assays may require additional validation.
• Prepare serial dilutions of the recombinant standard to cover the assay’s dynamic range (e.g., 2.8–2000 pg/mL).
• Include internal controls and matrix-matched standards if your sample buffer differs significantly from the standard diluent, to account for potential matrix effects.

Summary Table: Use of Recombinant Rat Fractalkine as ELISA Standard

Conclusion:
You can use recombinant Rat Fractalkine as a standard for ELISA quantification, provided it is validated, properly prepared, and recognized by your assay antibodies. Always follow best practices for standard preparation and assay validation to ensure accurate results.

Recombinant rat fractalkine (CX3CL1) has been validated for several important applications in published research:

Bioactivity and Functional Assays

The primary validated application is bioactivity assessment. Recombinant rat fractalkine has been specifically validated for its ability to chemoattract freshly isolated human peripheral blood lymphocytes (PBL), demonstrating its functional capacity as a chemotactic agent. This bioactivity validation is critical for confirming that the recombinant protein maintains its biological function comparable to the native molecule.

Neutralization Studies

Neutralization assays represent another validated application. This involves using recombinant rat fractalkine to evaluate the effectiveness of blocking antibodies or antagonists against the fractalkine-CX3CR1 axis. Such studies are particularly relevant for therapeutic development, as demonstrated by research examining humanized monoclonal antibodies targeting fractalkine in clinical contexts.

Western Blot Analysis

Western blot control applications have been validated. Recombinant rat fractalkine serves as a positive control and reference standard in Western blot experiments, allowing researchers to detect endogenous fractalkine expression in tissue samples and verify antibody specificity.

Research Applications

Beyond these standard validated applications, recombinant rat fractalkine has been employed in diverse research contexts, including studies on neurogenesis regulation, hematoma resolution and neuroprotection, microglial activation and polarization, and investigation of the fractalkine-CX3CR1 signaling pathway in various disease models. These applications leverage the protein's role in neuron-glia interactions and immune cell regulation.

Reconstitution Protocol

Recombinant rat fractalkine should be reconstituted in sterile, deionized water or phosphate-buffered saline (PBS) at a concentration of 100 µg/mL. Before opening the vial, briefly centrifuge it to bring the lyophilized powder to the bottom.

Add the appropriate volume of sterile buffer to the vial and allow the protein to dissolve gradually. Gentle agitation or mixing for 15-30 minutes at room temperature is recommended. If the powder does not fully dissolve initially and flakes remain visible, continue mixing for up to 2 hours at room temperature with gentle agitation.

Avoid vigorous shaking or vortexing, as this can denature the protein and reduce biological activity. Instead, gently swirl or blow the solution to facilitate dissolution.

Storage Conditions

Short-term storage: After reconstitution, store the protein at 2-8°C for up to one month. For immediate use in experiments, this temperature range is suitable for periods up to 2-7 days.

Long-term storage: For extended storage beyond one month, maintain the protein at -20°C to -70°C in a manual defrost freezer. Critically, avoid repeated freeze-thaw cycles, as these significantly compromise protein stability and biological activity.

To minimize freeze-thaw damage during long-term storage, prepare working aliquots of the reconstituted protein. Adding carrier proteins such as 0.1% bovine serum albumin (BSA), 10% fetal bovine serum (FBS), or 5% human serum albumin (HSA) can enhance stability. Additionally, incorporating 5-50% glycerol (final concentration) provides cryoprotection for storage at -20°C to -80°C.

Preparation for Cell Culture Applications

For cell culture experiments, further dilute the reconstituted stock solution to the desired working concentration using appropriate aqueous solutions. Optimal dilutions should be determined empirically for each specific application and laboratory setup.

The biological activity of rat fractalkine is typically determined by its ability to chemoattract immune cells, with specific activity ranging from 100,000-200,000 units/mg and an expected ED₅₀ of 0.003-0.009 µg/mL. For most applications, working concentrations of 5.0-10.0 ng/mL are commonly used.

Ensure all solutions, pipette tips, and equipment are sterile to maintain protein integrity and prevent contamination of your cell culture experiments. Handle the protein gently throughout all preparation steps to preserve its chemotactic and biological functions.