Recombinant Human MCP-2

Recombinant Human MCP-2 (monocyte chemoattractant protein-2, also designated CCL8) offers several compelling advantages for immunological and inflammatory research applications.

Biological Activity and Functional Specificity

Recombinant Human MCP-2 functions as a potent chemotactic factor with a unique receptor profile that distinguishes it from related chemokines. Unlike MCP-1, which primarily signals through CCR2B, MCP-2 demonstrates dual receptor functionality, binding to both CCR1 and CCR2B with high affinity. This dual-receptor engagement provides a broader spectrum of cellular responses compared to other C-C chemokines. Additionally, MCP-2 exhibits high-affinity binding to CCR5, a common HIV co-receptor, which has potential implications for studying HIV-related immune mechanisms.

Cellular Recruitment Capabilities

MCP-2 attracts a diverse array of leukocyte populations, including monocytes, lymphocytes, basophils, and eosinophils. This broad chemotactic range makes it particularly valuable for investigating inflammatory responses and immune cell recruitment. The protein induces dose-dependent chemotaxis and calcium mobilization in target cells, with significant biological activity observed at concentrations as low as 30-100 ng/mL. This sensitivity allows for precise experimental titration and detection of cellular responses.

Protein Characteristics and Quality

Recombinant Human MCP-2 is typically expressed in Escherichia coli as a non-glycosylated protein containing 76 amino acids with a molecular mass of approximately 8.9 kDa. Commercial preparations achieve high purity standards (>95%) with minimal endotoxin contamination (<0.1 EU/µg), ensuring reliable and reproducible experimental results. The protein is available in multiple formulations, including lyophilized powder and carrier-free preparations, accommodating various experimental protocols.

Research Applications

The protein is suitable for multiple analytical techniques including SDS-PAGE, HPLC, ELISA, and cell-based bioassays. Its ability to induce intracellular calcium mobilization and chemotactic responses makes it ideal for functional assays measuring immune cell activation. Furthermore, MCP-2 can bind heparin, which is relevant for studying interactions with extracellular matrix components and glycosaminoglycans in inflammatory microenvironments.

Unique Functional Properties

MCP-2 demonstrates unique functional properties compared to structurally similar chemokines despite sharing >60% sequence homology with MCP-1 and MCP-3. The processed N-terminal form, MCP-2(6-76), exhibits distinct regulatory functions, inhibiting the chemotactic effects of other chemokines including CCL7, CCL2, CCL5, and CCL8, providing opportunities to study chemokine cross-regulation.

Yes, recombinant human MCP-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative immunoassays, including ELISA, where recombinant proteins serve as standards to generate calibration curves for analyte quantification.

Key considerations and best practices:

• Purity and Characterization: The recombinant MCP-2 should be highly purified and well-characterized. Impurities or degradation products can affect the accuracy of your standard curve.
• Concentration Determination: The protein concentration must be accurately measured, typically by absorbance at 280 nm or another validated method, to ensure correct standard curve preparation.
• Matrix Compatibility: Prepare the standard curve in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate quantification.
• Reconstitution and Storage: If the recombinant MCP-2 is supplied lyophilized, follow the manufacturer’s reconstitution and storage instructions carefully to maintain protein integrity and activity.
• Validation: It is good practice to validate the recombinant standard in your specific ELISA system, confirming parallelism between the standard curve and endogenous MCP-2 in your sample matrix.

ELISA kits and protocols routinely use recombinant MCP-2 as a standard:

• Many commercial ELISA kits for MCP-2 quantification include a recombinant human MCP-2 standard, demonstrating its suitability for this purpose.
• Recombinant MCP-2 is also recommended for custom or in-house ELISA development, as long as the above criteria are met.

Caveats:

• Ensure the recombinant MCP-2 is from a reliable source and is suitable for use as an ELISA standard (not all recombinant proteins are validated for this purpose).
• If using a recombinant standard not provided with a kit, confirm that it matches the sequence and post-translational modifications (if relevant) of the native protein detected by your assay antibodies.

Summary Table:

In conclusion, recombinant human MCP-2 is widely accepted and used as a standard for ELISA quantification, provided it is properly prepared and validated for your assay system.

Recombinant Human MCP-2 (CCL8) has been validated for several applications in published research, primarily in functional assays (chemotaxis), SDS-PAGE, HPLC, ELISA, and as a standard or control in cell-based assays.

Key validated applications include:

• Chemotaxis assays: MCP-2 is widely used to assess its ability to attract monocytes, lymphocytes, basophils, eosinophils, and other immune cells. Its chemotactic activity is often measured using transwell migration assays or similar systems, including the chemoattraction of human PBMCs and mouse BaF/3 cells transfected with human CCR5.
• SDS-PAGE and HPLC: These analytical techniques are used to confirm the purity and integrity of the recombinant protein.
• ELISA (Enzyme-Linked Immunosorbent Assay): MCP-2 is used as a standard or positive control for quantifying MCP-2 levels in biological samples.
• Cell culture and functional studies: Recombinant MCP-2 is applied to cell cultures to study its effects on immune cell activation, migration, and signaling, particularly via receptors such as CCR1, CCR2B, and CCR5.
• Protein microarrays: MCP-2 has been included in protein microarrays for biomarker discovery and profiling cytokine responses in autoimmune and inflammatory diseases.

Additional context:

• MCP-2 is implicated in studies of allergic responses, inflammation, and neoplasia due to its role in immune cell recruitment and activation.
• The protein is also used in mechanistic studies to dissect chemokine-receptor interactions and to evaluate inhibitory effects of processed forms on chemotactic activity.

In summary, published research validates recombinant human MCP-2 for chemotaxis, analytical characterization (SDS-PAGE, HPLC), ELISA, cell-based functional assays, and protein microarrays. These applications support its use in immunology, inflammation, and biomarker discovery studies.

To reconstitute and prepare Recombinant Human MCP-2 (CCL8) protein for cell culture experiments, follow these steps for optimal solubility, stability, and biological activity:

1. Preparation Before Reconstitution

• Equilibrate the lyophilized protein vial and your chosen reconstitution buffer (typically sterile water or PBS) to room temperature.
• Centrifuge the vial briefly to collect all lyophilized powder at the bottom.

2. Reconstitution

• Buffer Choice: Use sterile, endotoxin-free water or PBS (pH 7.4) for initial reconstitution. For enhanced stability, especially at low concentrations, you may add 0.1% BSA as a carrier protein.
• Concentration: Reconstitute at a minimum of 100 μg/mL (0.1 mg/mL) for full solubility. Adjust volume based on the amount of protein in your vial.
• Technique: Gently pipette the buffer down the sides of the vial to ensure all protein is dissolved. Avoid vigorous mixing or vortexing, which can denature the protein.
• Incubation: Allow the solution to sit at room temperature for 10–30 minutes, gently swirling or inverting occasionally to facilitate dissolution.

3. Dilution for Cell Culture

• After reconstitution, dilute the stock solution to your desired working concentration using cell culture medium or buffer compatible with your assay. For chemotaxis assays, MCP-2 is typically used in the range of 30–500 ng/mL.
• If using serum-free conditions, avoid carrier proteins like BSA or FBS in your final dilution.

4. Storage

• Short-term: Store reconstituted protein at 2–8 °C for up to one week.
• Long-term: Aliquot and freeze at −20 °C to −80 °C to avoid repeated freeze-thaw cycles.
• For long-term storage, adding carrier proteins (e.g., 0.1% BSA) or stabilizers (e.g., 5–50% glycerol or trehalose) is recommended.

5. Handling Notes

• Always use sterile technique to avoid contamination.
• Avoid repeated freeze-thaw cycles by aliquoting into single-use volumes.
• If the protein does not dissolve completely, gently mix for up to 2 hours at room temperature.

Summary Table: Reconstitution Protocol

References for Best Practices:

If your experiment requires specific buffer conditions (e.g., serum-free, animal-free), adjust accordingly and consult the product’s Certificate of Analysis for any manufacturer-specific recommendations.