Recombinant Human MCP-1 (Mucin Stalk Chimera)

Using Recombinant Human MCP-1 (Mucin Stalk Chimera) in research applications is advantageous for studying chemokine-driven immune cell migration, inflammation, and disease mechanisms, as well as for bioactivity assays and cell signaling studies. The mucin stalk chimera format can enhance protein stability and mimic native glycosylation, improving experimental relevance.

Key scientific reasons to use this reagent:

• Potent chemotactic activity: MCP-1 (also known as CCL2) is a critical chemokine that regulates the migration and infiltration of monocytes, macrophages, and other immune cells, making it essential for studying immune cell trafficking, inflammation, and related pathologies.
• Bioactivity validation: The recombinant mucin stalk chimera form is validated for bioactivity, such as chemoattracting CCR2-expressing cells (e.g., BaF3 mouse pro-B cells transfected with human CCR2), ensuring functional relevance in assays.
• Disease modeling: MCP-1 is implicated in various diseases, including cancer, autoimmune disorders, infectious diseases, and neurodegenerative conditions, making it a valuable tool for modeling disease mechanisms and testing therapeutic interventions.
• Enhanced stability and mimicry of native protein: The mucin stalk chimera design can improve protein solubility, stability, and post-translational modifications, more closely resembling the native human protein and thus increasing the physiological relevance of in vitro and in vivo studies.
• Versatility in applications: Recombinant MCP-1 (Mucin Stalk Chimera) is suitable for a range of applications, including:
  • Chemotaxis and migration assays
  • Cell signaling and receptor activation studies
  • ELISA standards and controls
  • Functional studies in immunology, oncology, and neurobiology

Best practices:

• Use carrier-free formulations for sensitive cell-based assays to avoid interference.
• Select BSA-containing formulations for general cell culture or as ELISA standards to enhance protein stability.

In summary, Recombinant Human MCP-1 (Mucin Stalk Chimera) is a robust, bioactive reagent for dissecting chemokine biology, immune cell dynamics, and disease processes, with the mucin stalk chimera format offering improved stability and native-like properties for reliable experimental outcomes.

Yes, you can use recombinant human MCP-1 (Mucin Stalk Chimera) as a standard for quantification and calibration in ELISA assays. This recombinant protein is specifically recommended for use as an ELISA standard, particularly when formulated with BSA as a carrier protein.

Technical Considerations

Protein Characteristics

The recombinant human MCP-1 (Mucin Stalk Chimera) is a fusion protein consisting of human MCP-1 (CCL2, amino acids Gln24-Thr99) fused to the human Fractalkine mucin-like stalk domain, with a 6-His tag at the C-terminus. This construct has a molecular weight of approximately 35 kDa and is typically produced in mammalian expression systems (NS0-derived). The protein is supplied in lyophilized form from a 0.2 μm filtered solution in PBS with BSA as a carrier protein, with a purity of 97% as determined by SDS-PAGE under reducing conditions.

Standardization Protocol

When using this recombinant protein as a standard, it's important to follow established normalization practices. Standards should be validated in batch form, and subsequent preparations should be normalized to the original batch to ensure consistent unit definitions and lot-to-lot consistency. This approach maintains accuracy across multiple assay runs and different production lots.

Assay Performance

Standard colorimetric ELISA kits typically achieve sensitivity levels less than 10 pg/mL with detection ranges around 10-250 pg/mL, though some formulations offer wider ranges. The sandwich ELISA format used with MCP-1 detection provides good intra-assay precision (typically <10% coefficient of variation), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV).

This recombinant protein standard will provide reliable quantification when used according to established ELISA protocols with appropriate calibration curves and quality controls.

Recombinant Human MCP-1 (Mucin Stalk Chimera) has been validated primarily for bioactivity assays, specifically for its ability to induce chemotaxis in cells expressing the human CCR2 receptor.

Key validated applications in published research include:

• Chemotaxis Assays: The protein has been measured for its ability to chemoattract BaF3 mouse pro‑B cells transfected with human CCR2, demonstrating functional activity of the MCP-1 domain in the chimera.
• General Bioactivity: The recombinant chimera is routinely used in bioactivity assays to study MCP-1/CCL2-mediated signaling, migration, and receptor activation, confirming its utility in functional studies of chemokine-receptor interactions.

Supporting details:

• The mucin stalk chimera format is designed to mimic the membrane-tethered presentation of chemokines, which can be important for studying cell adhesion and migration under more physiologically relevant conditions.
• While the core application is chemotaxis, MCP-1/CCL2 is broadly used in research on monocyte/macrophage migration, inflammation, and immune cell trafficking. However, published validation for the mucin stalk chimera specifically centers on bioactivity and chemotaxis assays.

Additional relevant information:

• MCP-1/CCL2 is implicated in a wide range of biological processes, including immune surveillance, inflammation, and disease pathology such as cancer, atherosclerosis, and diabetes. The mucin stalk chimera format may be used to investigate these processes in vitro, but direct validation in published research is focused on chemotactic bioactivity.
• No published research currently validates this specific recombinant chimera for applications such as immunohistochemistry, ELISA, or in vivo studies; its use is primarily in functional cell-based assays.

In summary, the validated application for Recombinant Human MCP-1 (Mucin Stalk Chimera) in published research is bioactivity/chemotaxis assays using CCR2-expressing cells.

To reconstitute and prepare Recombinant Human MCP-1 (Mucin Stalk Chimera) protein for cell culture experiments, dissolve the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA).

Protocol and Best Practices:

• Reconstitution:

  • Add sterile PBS with ≥0.1% BSA directly to the lyophilized protein to achieve a final concentration of 100 μg/mL.
  • Gently mix or swirl the vial to ensure complete solubilization. Avoid vigorous vortexing, which may denature the protein.
  • If the protein appears as a film or does not dissolve immediately, allow it to sit at room temperature for several minutes and gently mix again.

• Dilution for Cell Culture:

  • After reconstitution, further dilute the stock solution to your desired working concentration using cell culture medium or PBS with 0.1% BSA. Typical working concentrations for functional assays range from 5–100 ng/mL, depending on the cell type and experimental design.
  • Always prepare fresh dilutions for each experiment to maintain protein activity.

• Storage:

  • Store the reconstituted protein at 2–8°C for up to one month or at –20°C to –70°C for long-term storage.
  • Avoid repeated freeze-thaw cycles, as these can denature the protein and reduce activity.
  • For long-term storage, aliquot the stock solution to minimize freeze-thaw events.

• Additional Notes:

  • The presence of BSA or HSA (≥0.1%) is critical for protein stability and to prevent adsorption to plasticware.
  • If the product contains a precipitate after reconstitution, centrifuge briefly to clarify before use.
  • Always use sterile technique to prevent contamination.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant MCP-1 for cell culture applications.