Recombinant Rat MCP-3

Recombinant Rat MCP-3 (CCL7) is a chemokine widely used in research to study immune cell migration, inflammation, and bone biology due to its potent chemotactic and immunomodulatory properties.

Key scientific reasons to use recombinant rat MCP-3 in research applications:

• Chemotaxis and Immune Cell Recruitment: MCP-3 is a strong chemoattractant for monocytes, eosinophils, and can activate NK cells and T lymphocytes (CD4+ and CD8+), making it essential for studies on immune cell trafficking, inflammation, and immune response modulation.

• Receptor Signaling: MCP-3 signals through multiple G protein-coupled receptors, including CCR1, CCR2, and CCR3, allowing investigation of receptor-specific pathways and cross-talk in immune and inflammatory processes.

• Bone Biology and Regeneration: Recombinant MCP-3 has been shown to promote osteoblast differentiation and function, increase bone formation, and inhibit osteoclast differentiation, making it valuable for research in bone metabolism, regeneration, and diseases such as osteoporosis.

• Inflammation and Disease Models: MCP-3 is produced by macrophages and tumor cell lines, and is involved in regulating macrophage function during inflammation and metastasis, supporting its use in models of inflammatory diseases, cancer, and tissue repair.

• Experimental Consistency: Recombinant MCP-3 provides a high-purity, biologically active, and consistent protein standard for cell biology assays, immunological studies, and as a calibrator in ELISA or other quantitative assays.

• Species-Specific Research: Using recombinant rat MCP-3 ensures species compatibility for in vitro and in vivo rat models, reducing cross-species variability and improving translational relevance for preclinical studies.

Typical applications include:

• Chemotaxis assays to study immune cell migration.
• Bone formation and resorption assays.
• Inflammatory disease and cancer models.
• ELISA standards and immunogen for antibody production.

Summary of best practices:

• Use recombinant rat MCP-3 for mechanistic studies of chemokine signaling, immune cell recruitment, and bone biology in rat models.
• Confirm biological activity and purity via chemotaxis bioassays and SDS-PAGE/HPLC analysis.
• Store protein aliquots at recommended temperatures to maintain stability and activity.

In conclusion, recombinant rat MCP-3 is a versatile tool for dissecting chemokine-mediated processes in immunology, inflammation, and bone research, providing reproducible and species-specific results for a wide range of experimental applications.

Yes, recombinant Rat MCP-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the native protein in immunoreactivity. Recombinant MCP-3 is commonly supplied as a calibrator or standard in commercial rat MCP-3 ELISA kits, where it is used to generate the standard curve for quantification of MCP-3 in biological samples.

Essential context and supporting details:

• Standard Curve Generation: ELISA kits for rat MCP-3 typically include lyophilized recombinant rat MCP-3 as the standard. The standard is reconstituted and serially diluted to create a calibration curve, which is then used to determine MCP-3 concentrations in unknown samples by comparison of their optical density (OD) values to the curve.
• Validation and Recovery: The use of recombinant MCP-3 as a standard is validated by recovery experiments, where known amounts of recombinant MCP-3 are spiked into matrices such as serum or plasma. Recovery rates in published protocols range from approximately 80% to 108%, indicating that recombinant MCP-3 behaves similarly to endogenous MCP-3 in these assays.
• Specificity: ELISA kits are designed to recognize both native and recombinant rat MCP-3, with no significant cross-reactivity to related chemokines, ensuring accurate quantification.
• Precision: Intra-assay and inter-assay coefficients of variation (CV) are typically below 10–15%, supporting the reliability of quantification using recombinant standards.
• Best Practices:
  • Ensure the recombinant MCP-3 standard is of high purity and properly quantified.
  • Reconstitute and dilute the standard according to the ELISA kit protocol.
  • Confirm that the recombinant MCP-3 sequence and post-translational modifications (if any) match those of the native rat MCP-3, as significant differences could affect antibody recognition and quantification accuracy.
  • Use freshly prepared standards and avoid repeated freeze-thaw cycles to maintain protein integrity.

Additional relevant information:

• Parallelism: It is good practice to verify parallelism between the standard curve generated with recombinant MCP-3 and the response of endogenous MCP-3 in your sample matrix. This ensures that the assay quantifies both forms equivalently.
• Documentation: Always refer to the ELISA kit manual for specific instructions regarding standard preparation and use, as protocols may vary between kits.

In summary, recombinant Rat MCP-3 is suitable for use as a standard in ELISA quantification, provided it is validated and matches the immunological properties of the native protein in your assay system.

Recombinant Rat MCP-3 (CCL7) has been validated for several applications in published research, primarily related to its biological activity and use as a research tool. Key applications include:

• Chemotaxis Assays: Recombinant Rat MCP-3 is widely used to study monocyte and leukocyte migration. Its ability to chemoattract monocytes, lymphocytes, basophils, and eosinophils has been validated in functional assays, often using concentrations in the range of 50–300 ng/mL.

• Inflammation and Immune Response Studies: MCP-3 is involved in regulating inflammation and macrophage function. It is used in research investigating inflammatory processes, including monocyte mobilization from bone marrow and recruitment to inflammatory sites.

• Fibrosis and Collagen Synthesis Research: Studies have shown that CCL7 (MCP-3) can stimulate type I collagen expression in skin fibroblasts, making it relevant for research on fibrosis and tissue remodeling.

• Protein Standard in Assays: Recombinant Rat MCP-3 serves as a protein standard in assays such as ELISA and Western blotting, where its purity and activity are critical.

• Immunogen: It is used as an immunogen for generating monoclonal and polyclonal antibodies, particularly in studies focused on chemokine signaling and immune cell regulation.

• Cell Biology Applications: The protein is employed in various cell biology studies, including investigations of chemokine receptor signaling (CCR1, CCR2, CCR3) and the regulation of macrophage function during inflammation and metastasis.

These applications are supported by its use in peer-reviewed research and its validation in functional and biochemical assays.

To reconstitute and prepare Recombinant Rat MCP-3 protein for cell culture experiments, first centrifuge the vial briefly, then add sterile water or buffer to achieve a concentration of 0.1–1.0 mg/mL, gently mix without vortexing, and further dilute in cell culture medium or buffer containing carrier protein (e.g., 0.1% BSA) as needed for your assay.

Step-by-step protocol:

• Centrifuge the vial briefly (e.g., 1 min at 1000 × g) before opening to ensure all lyophilized powder is at the bottom.
• Add sterile water or buffer:
  • Most protocols recommend reconstituting in sterile distilled water or sterile PBS, pH 7.2–7.4.
  • Typical starting concentration is 0.1 mg/mL (100 µg/mL) or higher, depending on your experimental needs.
• Gently mix:
  • Pipette up and down gently and wash down the sides of the vial to ensure complete dissolution.
  • Do not vortex; avoid vigorous shaking to prevent protein denaturation.
  • Allow the protein to dissolve for 10–30 minutes at room temperature. If visible particulates remain, continue gentle mixing for up to 2 hours.
• Dilute for cell culture:
  • For cell culture applications, further dilute the reconstituted stock in your cell culture medium or buffer containing a carrier protein such as 0.1% BSA or 10% FBS to stabilize the protein and minimize adsorption to plasticware.
• Aliquot and storage:
  • Aliquot the reconstituted protein into working volumes to avoid repeated freeze-thaw cycles.
  • Store at 4°C for up to 1 week; for long-term storage, keep aliquots at –20°C to –80°C.
  • For extended storage, adding 5–50% glycerol can help maintain protein stability.
• Avoid repeated freeze-thaw cycles to preserve protein activity.

Additional notes:

• Always check the specific datasheet for your batch for recommended buffer and concentration.
• If using for chemotaxis or signaling assays, typical working concentrations range from 50–300 ng/mL.
• If the protein was lyophilized from PBS, reconstitution in PBS is preferred; otherwise, use sterile water or the buffer specified in the datasheet.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant Rat MCP-3 for cell culture experiments.