Recombinant Human MIP-5 (CCL15) (68 aa)

Recombinant Human MIP-5 (CCL15) (68 aa) is a truncated, biologically active form of the chemokine CCL15, widely used in research to study immune cell recruitment, inflammatory signaling, and chemokine-receptor interactions.

Key scientific reasons to use this protein in your research applications:

• Chemotaxis Assays: CCL15 (MIP-5) is a potent chemoattractant for monocytes, T cells, neutrophils, and lymphocytes. It enables quantitative studies of immune cell migration, which is fundamental for immunology, inflammation, and cancer research.

• Receptor Signaling Studies: CCL15 binds primarily to CCR1 (high affinity) and CCR3 (lower affinity), activating downstream signaling pathways such as Gi/Go protein signaling, phospholipase C, PKCδ, and NF-κB. This makes it valuable for dissecting chemokine-receptor interactions and intracellular signaling mechanisms.

• Inflammatory Disease Models: CCL15 is implicated in leukocyte recruitment and the development of inflammatory diseases, including asthma, atherosclerosis, and sarcoidosis. Using recombinant CCL15 allows you to model and manipulate inflammatory responses in vitro and in vivo.

• High Purity and Defined Sequence: The 68 aa recombinant form is highly purified (typically >97% by HPLC/SDS-PAGE) and sequence-defined, ensuring reproducibility and specificity in experimental setups.

• Functional Activity: The recombinant 68 aa CCL15 is biologically active, as demonstrated by its ability to induce chemotaxis in human T-lymphocytes and monocytes at low nanogram concentrations. This ensures reliable results in functional assays.

• Versatility in Immunology Research: CCL15 is expressed in T and B lymphocytes, NK cells, monocytes, dendritic cells, and airway smooth muscle cells. Its broad expression profile allows investigation into diverse aspects of immune regulation and cell signaling.

• Experimental Control: Recombinant proteins provide precise control over concentration, purity, and formulation, which is critical for dose-response studies, mechanistic experiments, and reproducibility.

Typical applications include:

• Chemotaxis and migration assays
• Receptor binding and signaling studies
• In vitro modeling of inflammatory responses
• Cell activation and cytokine release assays
• Studies of disease mechanisms involving immune cell recruitment

Best practices:

• Reconstitute in sterile water at recommended concentrations (≥100 μg/ml), dilute as needed, and store aliquots at −18°C with carrier protein to prevent freeze-thaw cycles.
• Use in strictly controlled laboratory research settings; not for therapeutic or diagnostic use.

In summary:
Using recombinant human MIP-5 (CCL15) (68 aa) enables precise, reproducible investigation of chemokine biology, immune cell migration, and inflammatory signaling, making it a valuable tool for immunology, cell biology, and disease modeling research.

You can use recombinant human MIP-5 (CCL15) (68 aa) as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and its sequence matches the region recognized by the antibodies in your ELISA kit.

Key considerations:

• Purity and Quantification: The recombinant protein should be highly purified (typically ≥98% by SDS-PAGE/HPLC) and its concentration must be accurately known to prepare reliable standard curves.
• Sequence Compatibility: Many commercial ELISA kits for CCL15/MIP-5 use truncated forms (e.g., CCL15(22-92), CCL15(25-92), or CCL15(29-92)), which are more potent chemoattractants and may be the immunogen for the capture/detection antibodies. If your recombinant protein is the 68 amino acid form, confirm that its sequence overlaps with the region recognized by the kit antibodies. If the antibodies target epitopes outside the 68 aa region, the standard may not be detected equivalently.
• Standard Curve Preparation: Follow best practices for standard curve preparation, including proper reconstitution, dilution, and use of the same buffer matrix as your samples to minimize matrix effects.
• Validation: It is recommended to validate the recombinant standard by running a parallel standard curve with the kit’s supplied standard (if available) to ensure comparable detection and quantification.

Limitations:

• If the ELISA kit is validated with a different isoform or length of CCL15 (e.g., full-length 92 aa), the 68 aa recombinant may not be recognized identically, potentially affecting quantification accuracy.
• Always check the ELISA kit datasheet or contact the manufacturer for information on the immunogen and recommended standard protein length.

Summary Table:

In summary, you can use the 68 aa recombinant MIP-5 as a standard for ELISA quantification if it meets the above criteria, but sequence compatibility and validation are critical for accurate results.

Recombinant Human MIP-5 (CCL15) (68 aa) has been validated for several key applications in published research, primarily in the context of immunology and cell signaling studies.

Validated Applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or control to quantify CCL15/MIP-5 levels in biological samples.
• Functional Assays: Demonstrated activity in chemotaxis assays, particularly for attracting human T lymphocytes and monocytes, confirming its biological function as a chemokine.
• Western Blot: Used as a positive control or for antibody validation in protein detection experiments.

Supporting Details:

• The protein is commonly used to study chemotactic responses of immune cells, especially via its interaction with the CCR1 receptor.
• Research has implicated MIP-5/CCL15 in the pathogenesis of diseases such as asthma, sarcoidosis, and atherosclerosis, and in the regulation of stem cell proliferation, suggesting its use in disease model systems and mechanistic studies.
• The 68 amino acid form is a truncated, biologically active version, often used in these assays for its robust chemotactic activity.

Additional Notes:

• Most commercial and research-use preparations are validated for the above applications, but specific published studies should be consulted for detailed protocols and experimental outcomes.
• The protein is typically supplied as a lyophilized powder, expressed in E. coli, and is reconstituted for use in aqueous buffers with carrier proteins to maintain stability and activity.

Summary Table:

If you require information on a specific disease model or experimental protocol involving this protein, please specify for a more targeted literature review.

To reconstitute and prepare Recombinant Human MIP-5 (CCL15) (68 aa) for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water to achieve a final concentration between 0.1–1.0 mg/mL. Avoid concentrations above 1 mg/mL, as this may affect solubility and stability.

Step-by-step protocol:

• Before opening the vial: Briefly centrifuge the lyophilized protein vial (20–30 seconds, microcentrifuge) to ensure all powder is at the bottom.
• Reconstitution: Add sterile, distilled water to the vial to reach your desired concentration (e.g., for 100 µg protein, add 100 µL for 1 mg/mL or 1 mL for 0.1 mg/mL).
• Mixing: Gently swirl or tap the vial to dissolve. Do not vortex or shake vigorously, as this can denature the protein.
• Incubation: If solubility is an issue, allow the solution to incubate at 4°C overnight.
• Carrier protein: For working solutions and long-term storage, dilute further in an aqueous buffer containing 0.1–1.0% BSA or HSA to prevent adsorption and loss, especially at concentrations below 0.1 mg/mL.
• Aliquoting: Divide the reconstituted protein into small aliquots to avoid repeated freeze-thaw cycles.
• Storage:
  • Short-term (2–7 days): Store at 4°C.
  • Long-term: Store aliquots at ≤–20°C (preferably –80°C) with carrier protein.
  • Avoid repeated freeze-thaw cycles, which can cause denaturation.
• Quality check: Confirm protein recovery and integrity by SDS-PAGE if needed (a band at expected size should be visible with as little as 10 ng loaded).

Additional notes:

• Use low-binding tubes to minimize protein loss due to adsorption.
• Prepare working dilutions fresh before each experiment, especially if at low concentrations (<0.1 mg/mL).
• Always consult the product-specific datasheet for buffer recommendations, as some proteins may require low pH or specific additives for optimal solubility.

This protocol ensures maximal recovery, stability, and biological activity of recombinant MIP-5 (CCL15) for cell culture applications.