Recombinant Human MCP-1

Recombinant Human MCP-1 (CCL2) is widely used in research applications because it is a key chemokine that regulates the migration and infiltration of monocytes and macrophages, making it essential for studies of inflammation, immune cell trafficking, and disease mechanisms.

Key scientific applications and rationale:

• Chemotaxis assays: MCP-1 is a potent chemoattractant for monocytes, macrophages, and other immune cells. Using recombinant MCP-1 allows precise control of experimental conditions to study cell migration, recruitment, and signaling pathways in vitro.
• Inflammation and immunology research: MCP-1 is upregulated in response to tissue injury and infection, and is involved in the extravasation of immune cells across blood vessel walls. It is critical for modeling inflammatory responses, immune surveillance, and the pathogenesis of diseases such as rheumatoid arthritis, atherosclerosis, and multiple sclerosis.
• Cancer biology: MCP-1 plays a complex role in tumor development by recruiting monocytes and macrophages to the tumor microenvironment, promoting angiogenesis, and modulating immune responses. Recombinant MCP-1 is used to dissect these mechanisms and evaluate therapeutic strategies targeting the MCP-1/CCR2 axis.
• Cell differentiation studies: MCP-1 regulates the differentiation of monocytes into dendritic cells, macrophages, and osteoclasts, making it valuable for studies on immune cell development and function.
• Neuroscience: MCP-1 is implicated in neurogenesis, neuroprotection, and neurotransmission, supporting its use in models of neuroinflammation and neurodegenerative diseases.
• Bioassays and functional studies: Recombinant MCP-1 is used in bioassays to study receptor-ligand interactions, signaling pathways, and cellular responses, including migration, proliferation, and differentiation.

Technical advantages:

• Consistency and reproducibility: Recombinant MCP-1 provides a defined, consistent source of protein, minimizing variability compared to native or serum-derived MCP-1.
• Compatibility with multiple assay formats: It can be used in chemotaxis assays, ELISA standards, Western blot controls, and cell culture experiments.
• Stability: Recombinant MCP-1 is stable under standard laboratory storage conditions, facilitating reliable experimental workflows.

Summary of best practices:

• Use recombinant MCP-1 for controlled studies of immune cell migration, inflammation, and cell differentiation.
• Select appropriate concentrations and assay formats based on your experimental design and cell type.
• Validate MCP-1 activity in your system using positive controls and functional assays.

In summary, recombinant human MCP-1 is essential for mechanistic studies of immune cell trafficking, inflammation, cancer biology, and cell differentiation, offering reproducibility and versatility across a wide range of research applications.

You can use recombinant human MCP-1 as a standard for quantification or calibration in your ELISA assays, provided it is properly validated and compatible with your assay system.

Key considerations and supporting details:

• Recombinant MCP-1 is commonly used as a standard in commercial ELISA kits for human MCP-1 (also known as CCL2). These kits often calibrate their standard curves using highly purified recombinant human MCP-1, typically expressed in E. coli or other systems.
• Parallelism and equivalence: Data from validated kits show that recombinant MCP-1 produces standard curves that are parallel to those generated with natural MCP-1, indicating that quantification is accurate for both recombinant and natural forms.
• Validation: Ensure that the recombinant MCP-1 you use is of high purity, has a defined concentration, and is compatible with the antibodies in your ELISA. Some kits specify that their standards are calibrated against international or in-house reference preparations.
• Matrix effects: For best accuracy, dilute your recombinant MCP-1 standard in the same buffer or matrix as your samples to minimize matrix effects and ensure comparable binding and detection.
• Documentation: Refer to your ELISA kit’s manual for recommended standard preparation and dilution protocols. Many kits provide detailed instructions for generating a standard curve using recombinant MCP-1.

Best practices:

• Use recombinant MCP-1 from a reliable source, with a certificate of analysis confirming its concentration and purity.
• Prepare serial dilutions as described in your ELISA protocol to generate a standard curve covering the expected range of your samples.
• If possible, verify that your recombinant standard yields a linear, parallel response compared to the kit’s supplied standard or to natural MCP-1, especially if using a custom or in-house assay.

In summary, recombinant human MCP-1 is suitable as a standard for ELISA quantification, provided it is validated for your specific assay conditions and antibody pairings.

Recombinant human MCP-1 (monocyte chemoattractant protein-1, also known as CCL2) has been validated for several key applications in published research:

Bioassay Applications

The primary application of recombinant MCP-1 is in bioassay studies examining its chemotactic and biological functions. Specifically, recombinant MCP-1 has been used to demonstrate dose-dependent monocyte migration and chemotaxis. The protein stimulates dose-dependent proliferation of monocytic cell lines such as THP-1 cells and chemoattracts transfected cell lines expressing the CCR2 receptor (such as BaF3 cells transfected with human CCR2A) in a dose-dependent manner, with effective doses ranging from 0.3–1.8 ng/mL.

ELISA Applications

Recombinant MCP-1 has been validated as an ELISA standard and control antigen. It has been successfully employed as a calibrator for assay standardization and as a control antigen in the development of sensitive ELISA assays using capture antibodies.

Neutralization Studies

The protein has been used in neutralization experiments where MCP-1-neutralizing antibodies are employed to block its biological activity, demonstrating the mediating role of MCP-1 in monocyte migration and inflammatory responses.

Western Blot Control

Recombinant MCP-1 serves as a Western blot control for validating detection methods and antibody specificity.

Research Applications

Beyond these standard applications, recombinant MCP-1 has been utilized in studies investigating monocyte migration, macrophage polarization, immune cell infiltration, and inflammatory disease mechanisms, including research on atherosclerosis, rheumatoid arthritis, and multiple sclerosis.

To reconstitute and prepare Recombinant Human MCP-1 protein for cell culture experiments, dissolve the lyophilized protein in sterile distilled water or PBS, typically to a concentration of 0.1–0.5 mg/mL (100–500 μg/mL), then dilute to your desired working concentration in cell culture medium containing carrier protein such as 0.1% BSA.

Step-by-step protocol:

• Before opening the vial: Briefly centrifuge to collect all material at the bottom.
• Reconstitution: Add sterile distilled water or PBS (check product datasheet for recommended buffer) to achieve a stock concentration of 0.1–0.5 mg/mL. For example, if the vial contains 10 μg, add 100 μL for a 0.1 mg/mL solution.
• Mixing: Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking to prevent foaming and protein denaturation.
• Incubation: Allow the protein to fully dissolve for 10–30 minutes at room temperature.
• Aliquoting: Divide the stock solution into small aliquots to avoid repeated freeze-thaw cycles, which can reduce protein stability.
• Storage: Store aliquots at ≤–20°C. For long-term storage, –70°C is preferred. Avoid repeated freeze-thaw cycles.
• Dilution for cell culture: Prepare working solutions (typically in the ng/mL range) by diluting the stock in cell culture medium or PBS containing 0.1% BSA or other carrier protein to minimize adsorption and loss.
• Use: Add the working solution directly to your cell culture as required by your experimental design.

Additional notes:

• Carrier protein (e.g., BSA): Including 0.1% BSA in dilution buffers helps stabilize MCP-1 and prevents loss due to adsorption to plasticware.
• Stability: MCP-1 is stable at working concentrations for at least 24 hours under standard cell culture conditions, but for longer storage, keep at –20°C or lower.
• Verification: You may confirm reconstitution by SDS-PAGE, loading as little as 10 ng to visualize the protein band.

Typical working concentrations: For cell-based assays, MCP-1 is often used at 5–30 ng/mL, but optimal concentrations depend on your specific cell type and assay.

Summary Table:

Always consult the specific product datasheet for any manufacturer-specific recommendations regarding buffer composition and reconstitution volume.