Recombinant Human CX3CL1/Fractalkine Protein

Recombinant Human CX3CL1/Fractalkine Protein is used in research to study immune cell migration, adhesion, neuroinflammation, and disease mechanisms involving the CX3CL1/CX3CR1 axis. Its unique dual function as both a soluble chemoattractant and a membrane-bound adhesion molecule makes it a valuable tool for dissecting cell signaling and immune responses in various physiological and pathological contexts.

Key scientific applications and rationale include:

• Modeling Chemotaxis and Cell Adhesion:
  The soluble form of CX3CL1 acts as a potent chemoattractant for T cells and monocytes, enabling in vitro and in vivo studies of immune cell migration. The membrane-bound form promotes strong adhesion of leukocytes to activated endothelial cells, allowing investigation of leukocyte trafficking and vascular inflammation.

• Neuroinflammation and Neuroprotection:
  CX3CL1 is constitutively expressed by neurons and modulates microglial activation, making it essential for research on neuroinflammatory diseases, neurodegeneration, and CNS injury models. It can be used to study neuron-microglia interactions and the balance between neurotoxic and neuroprotective immune responses.

• Disease Mechanism Elucidation:
  The CX3CL1/CX3CR1 axis is implicated in a range of diseases, including atherosclerosis, rheumatoid arthritis, cancer, and HIV infection. Recombinant CX3CL1 enables mechanistic studies of its role in inflammation, tumor immunity, and tissue regeneration.

• Therapeutic Target Validation:
  By blocking or stimulating the CX3CL1/CX3CR1 pathway with recombinant protein, researchers can evaluate its potential as a therapeutic target in models of autoimmune disease, cardiovascular disease, and cancer.

• Functional Assays and Controls:
  Recombinant CX3CL1 is used in bioassays to test chemotactic responses, adhesion, and signaling in CX3CR1-expressing cells, and as a control in ELISA or neutralization experiments.

• Cell Differentiation Studies:
  It regulates differentiation of monocytes and dendritic cells, supporting research into osteoclastogenesis and immune cell development.

Summary of advantages:

• High specificity for CX3CR1-expressing cells.
• Enables controlled, reproducible experiments.
• Facilitates both mechanistic and translational research across immunology, neuroscience, and oncology.

In summary, using recombinant human CX3CL1/Fractalkine protein allows precise manipulation of the CX3CL1/CX3CR1 axis, supporting diverse research applications from basic cell biology to disease modeling and therapeutic development.

Yes, recombinant human CX3CL1/Fractalkine protein can be used as a standard for quantification and calibration in ELISA assays. This is a well-established practice in the field.

Standard Protein Characteristics

Recombinant human CX3CL1/Fractalkine standards are typically expressed in mammalian cell systems, such as NS0 cells, which provide appropriate post-translational modifications and protein folding. The standard protein used in commercial ELISA kits represents the soluble chemokine portion of Fractalkine (residues 25-100 of the full protein sequence), which is the biologically active form generated through proteolytic cleavage from the transmembrane precursor.

Validation and Performance

The recombinant protein standards have been validated for accurate quantitation of both recombinant and natural Fractalkine. Studies demonstrate that results obtained using natural Fractalkine show linear curves that are parallel to the standard curves generated using recombinant protein standards, indicating reliable cross-reactivity and measurement accuracy. This parallel linearity is a critical validation criterion confirming that the recombinant standard appropriately represents endogenous protein in biological samples.

Practical Considerations

When using recombinant CX3CL1/Fractalkine as a calibration standard, ensure that:

• The standard is prepared in the appropriate calibrator diluent recommended by your assay protocol
• Samples are diluted appropriately to fall within the dynamic range of your standard curve
• The assay recognizes both natural and recombinant forms of the protein, which is standard for validated ELISA kits

The sensitivity and dynamic range of your assay will depend on the specific antibody pairs and detection method employed, with typical sensitivities ranging from approximately 1-300 pg/mL depending on the kit configuration.

Recombinant Human CX3CL1/Fractalkine Protein has been validated for several key applications in published research, primarily involving its roles as a chemoattractant, adhesion molecule, and modulator of immune cell function.

Validated Applications in Published Research:

• Bioassays:
  Used to assess chemotactic activity for T cells and monocytes. The soluble chemokine domain of human CX3CL1 has been shown to induce migration of these immune cells in vitro, confirming its bioactivity in chemotaxis assays.

• Cell Adhesion Studies:
  The membrane-bound form of CX3CL1 has been validated for promoting leukocyte adhesion to endothelial cells, both in static and flow-based adhesion assays.

• Western Blot Control:
  Recombinant human CX3CL1 has been used as a positive control in Western blotting to detect endogenous or recombinant protein expression and to validate antibody specificity.

• Neutralization Assays:
  Employed in experiments where neutralizing antibodies or inhibitors are tested for their ability to block CX3CL1-mediated effects, such as chemotaxis or adhesion.

• Binding Assays:
  Utilized to study receptor-ligand interactions (e.g., with CX3CR1) in binding assays, including quantitative analysis of binding kinetics and affinity.

• Functional Studies in Disease Models:
  Recombinant CX3CL1 has been administered in vivo or in vitro to study its effects on:

  • Monocyte and dendritic cell differentiation (e.g., into osteoclasts).
  • Macrophage chemotaxis and efferocytosis (clearance of apoptotic cells).
  • Neuroprotection and microglial regulation in CNS injury and neurodegeneration models.
  • Tumor suppression and immune cell recruitment in cancer models.

Summary Table of Validated Applications

Additional Notes:

• Both soluble and membrane-bound forms have been used, depending on the biological question.
• Applications span in vitro (cell-based) and in vivo (animal model) studies, including direct administration of recombinant protein to validate physiological effects.
• The protein is also used as a tool to dissect signaling pathways and immune cell trafficking in inflammation, neurobiology, and oncology research.

If you require protocols or more specific details for a particular application, please specify the context or experimental system.

To reconstitute and prepare Recombinant Human CX3CL1/Fractalkine Protein for cell culture experiments, follow these steps:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Reconstitution buffer:
   • If the protein is lyophilized without carrier protein, use sterile distilled water or sterile PBS.
   • If the protein is lyophilized with BSA as a carrier, use sterile PBS containing at least 0.1% human or bovine serum albumin to minimize adsorption and loss.
3. Concentration:
   • Common reconstitution concentrations are 0.1–1.0 mg/mL in sterile distilled water, or 25–100 μg/mL in sterile PBS (with or without 0.1% BSA, depending on formulation).
4. Dissolve gently: Mix by gentle pipetting or inversion. Avoid vigorous vortexing to prevent protein denaturation.
5. Aliquot and storage:
   • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles.
   • Store at 2–8°C for up to 1 week for short-term use, or at –20°C to –80°C for long-term storage.
6. Working solution:
   • For cell culture, dilute the stock to the desired working concentration (typically in the ng/mL to low μg/mL range) using cell culture medium or assay buffer immediately before use.

Additional notes:

• If your application is sensitive to endotoxin, confirm the endotoxin level is suitable for cell culture (<1 EU/μg is typical for most applications).
• Always use aseptic technique to prevent contamination.
• If the protein is supplied in a liquid formulation (e.g., PBS with glycerol), it can be used directly after dilution to the desired concentration.

Summary Table:

These guidelines are broadly applicable, but always check the specific product datasheet for any unique instructions.