Recombinant Human RANTES (Mucin Stalk Chimera)

Recombinant Human RANTES (Mucin Stalk Chimera) offers several compelling advantages for research applications, particularly in immunology, inflammation, and cell biology studies.

Enhanced Stability and Functionality

The mucin-like stalk chimera design provides superior characteristics compared to standard RANTES preparations. In this construct, the chemokine domain of human RANTES (CCL5) is fused to a mucin-like stalk derived from Fractalkine, creating a protein that maintains high biological activity while offering improved stability in experimental conditions. This architectural modification allows the protein to function as a ligand for CCR1, CCR3, and CCR5 receptors with demonstrated potency, with effective concentrations (ED50) ranging from 0.1–0.5 µg/mL in cell-based assays.

Broad Immunological Applications

RANTES is a potent chemoattractant capable of recruiting multiple leukocyte populations to inflammatory sites, including T cells, macrophages, eosinophils, and basophils. This makes it invaluable for studying immune cell recruitment, migration, and activation. The protein demonstrates particular utility in investigating delayed-type hypersensitivity reactions, allergic responses, and chronic inflammatory conditions such as rheumatoid arthritis and asthma.

Research Versatility

The recombinant form is suitable for multiple experimental methodologies, including functional assays, ELISA, Western blotting, immunohistochemistry, and flow cytometry applications. The protein is typically supplied with high purity (>90% by SDS-PAGE) and low endotoxin levels (<1.0 EU/mg), ensuring reliable and reproducible results across different experimental platforms.

Disease Model Applications

RANTES plays significant roles in angiogenesis, lymphogenesis, and immune modulation within tumor microenvironments, making it particularly valuable for cancer biology research. Additionally, the protein's involvement in viral infections and various inflammatory pathologies makes it suitable for studying disease mechanisms and testing therapeutic interventions.

The mucin stalk chimera format specifically provides a standardized, well-characterized reagent that combines the biological activity of native RANTES with enhanced experimental reliability and consistency.

You can use recombinant human RANTES (Mucin Stalk Chimera) as a standard for quantification or calibration in ELISA assays, provided certain conditions are met, but there are important caveats regarding its suitability depending on your assay design and the nature of the chimera.

Key considerations:

• Protein Structure: The RANTES (Mucin Stalk Chimera) is a fusion protein where the chemokine domain of human RANTES (CCL5) is fused to the mucin-like stalk of human Fractalkine. This means it is not identical to native RANTES; the additional mucin stalk may alter its conformation, epitope accessibility, or antibody recognition compared to native or full-length recombinant RANTES.

• ELISA Antibody Specificity: Most commercial ELISA kits for RANTES/CCL5 are designed to detect the native form of the protein. If your ELISA uses antibodies that specifically recognize epitopes present only in the native RANTES and not in the chimera, the standard curve generated with the chimera may not accurately reflect the concentration of native RANTES in your samples. This could lead to under- or overestimation of analyte levels.

• Validation: If you intend to use the mucin stalk chimera as a standard, you must validate that your ELISA antibodies recognize the chimera with the same efficiency as the native protein. This involves running parallel standard curves with both the chimera and a native RANTES standard, and comparing their slopes and detection ranges. If the curves are parallel and overlapping, the chimera can be used as a standard. If not, quantification will not be accurate.

• Best Practices: For quantitative ELISA, it is generally recommended to use a standard that matches the analyte in your samples as closely as possible in terms of sequence and post-translational modifications. This ensures the standard curve accurately reflects the behavior of the analyte in your assay.

• Manufacturer Recommendations: Some recombinant protein suppliers note that their RANTES (Mucin Stalk Chimera) can be used as an ELISA standard, especially if formulated with BSA for stability. However, this is contingent on compatibility with your specific ELISA antibodies and protocol.

Summary Table:

Recommendations:

• Validate the chimera as a standard in your specific ELISA by comparing its standard curve to that of native RANTES.
• If your ELISA is designed for native RANTES and you cannot validate the chimera, use a standard that matches the native sequence.
• Always check the ELISA kit documentation for recommended standards and compatibility notes.

If you provide more details about your ELISA (e.g., antibody specificity, kit type), more tailored guidance can be given.

Recombinant Human RANTES (Mucin Stalk Chimera) has been validated in published research primarily for applications involving immunization, antibody generation, and functional studies of chemokine activity.

Key validated applications include:

• Immunization for Nanobody Generation:
  The recombinant CCL5/RANTES (Mucin Stalk Chimera) has been used as an immunogen to generate and characterize nanobodies (single-domain antibodies) targeting CCL5/RANTES. In a published study, this protein was included in a mixture of carrier-free human recombinant chemokines (including the mucin stalk chimera forms) used to immunize animals for the production of neutralizing nanobodies against inflammatory chemokines such as CCL2, CCL5, and CXCL11. The resulting nanobodies were then validated for their ability to neutralize chemokine activity in vitro and in vivo.

• Functional Assays:
  The same study validated the use of the recombinant CCL5/RANTES (Mucin Stalk Chimera) in functional assays to assess the neutralizing capacity of generated nanobodies. These assays included chemotaxis inhibition and other bioactivity measurements relevant to chemokine function.

• Potential Use in Immunoassays and ELISA:
  While not directly cited in the primary research article, recombinant chemokines—including mucin stalk chimeras—are commonly used as standards or targets in immunoassays such as ELISA, flow cytometry, and other immunological assays to study chemokine-receptor interactions and immune cell migration. This is consistent with general best practices for recombinant chemokine use in research.

Additional Context:

• The mucin stalk chimera format is designed to enhance protein stability and mimic the presentation of chemokines on cell surfaces, which can be important for studying receptor binding and immune modulation in a more physiologically relevant context.
• Recombinant chemokines, including RANTES/CCL5, are also widely used in studies of immune cell recruitment, inflammation, and as adjuvants in vaccine research, though specific validation of the mucin stalk chimera format for these applications should be confirmed in each case.

Summary Table:

If you require details on a specific application or protocol, please specify the context (e.g., cell migration, receptor binding, vaccine adjuvant studies).

To reconstitute and prepare Recombinant Human RANTES (Mucin Stalk Chimera) protein for cell culture experiments, dissolve the lyophilized protein at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). This carrier protein helps stabilize the chemokine and prevents adsorption to plasticware.

Essential steps and considerations:

• Use sterile technique throughout the process to avoid contamination.
• Gently add the appropriate volume of sterile PBS + 0.1% BSA to the vial. Do not vortex; instead, gently pipette up and down to dissolve the protein completely.
• Allow the solution to sit at room temperature for a few minutes to ensure full dissolution.
• After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Store aliquots at 2–8°C for up to 1 month or at –20°C to –70°C for long-term storage. Avoid repeated freeze-thaw cycles.
• For cell culture experiments, dilute the stock solution to the desired working concentration using cell culture medium or buffer containing at least 0.1% BSA to maintain protein stability.

Additional recommendations:

• The final working concentration for cell assays typically ranges from 1–100 nM, depending on the experimental design and cell type.
• Always include appropriate controls, such as vehicle-only and untreated cells, to account for any effects of the buffer or carrier protein.
• If using the protein for chemotaxis or signaling assays, pre-equilibrate the working solution to the assay temperature before adding to cells.

Summary Table: Reconstitution Protocol

This protocol ensures optimal stability and biological activity of the recombinant RANTES (Mucin Stalk Chimera) for cell culture experiments.