Recombinant Human Macrophage Inflammatory Protein-5 (MIP-5)

Recombinant Human Macrophage Inflammatory Protein-5 (MIP-5) is primarily used in research applications to study immune cell chemotaxis, inflammatory signaling, and the regulation of immune responses, especially involving T-lymphocytes and macrophages.

Key scientific applications and rationale:

• Chemotaxis Assays: MIP-5 is a potent chemoattractant for human T-lymphocytes, making it valuable for in vitro migration assays to investigate immune cell trafficking and recruitment mechanisms. Significant chemotactic activity is observed at concentrations as low as 1–10 ng/mL.

• Immune Modulation Studies: As a member of the CC chemokine family, MIP-5 plays a role in modulating inflammatory responses by recruiting and activating leukocytes (including monocytes, T cells, and macrophages) to sites of inflammation. This is critical for dissecting the molecular pathways of inflammation and immune regulation.

• Macrophage Polarization and Function: MIP-5 can be used to study macrophage activation, polarization, and their role in inflammatory diseases. It is relevant for research into innate immunity, tissue homeostasis, and the response to pathogens or injury.

• Receptor-Ligand Interaction Studies: MIP-5 interacts with specific chemokine receptors (e.g., CCR1, CCR5), enabling research into receptor expression, signaling pathways, and downstream effects in hematopoietic and immune cells.

• Disease Models: MIP-5 is expressed at high levels in tissues such as liver, intestine, and lung leukocytes, making it useful for modeling tissue-specific immune responses and studying diseases characterized by inflammation or immune cell infiltration.

Best practices for use:

• Employ recombinant MIP-5 in controlled in vitro assays to ensure reproducibility and specificity.
• Use appropriate concentrations (typically 1–10 ng/mL for chemotaxis) based on published protocols.
• Confirm biological activity by measuring chemotactic response or downstream cytokine production in target cells.

Summary:
Use recombinant human MIP-5 in your research to elucidate mechanisms of immune cell migration, inflammatory signaling, and macrophage function, which are central to understanding and manipulating immune responses in health and disease.

You can use recombinant human Macrophage Inflammatory Protein-5 (MIP-5) as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with the antibodies used in your assay.

Key considerations and supporting details:

• ELISA kits for MIP-5 typically use recombinant protein as the standard. The standard provided in commercial kits is usually a recombinant form of human MIP-5, reconstituted and serially diluted to generate a standard curve for quantification. This standard is used to interpolate the concentration of MIP-5 in unknown samples by comparing their optical density (OD) values to the standard curve.

• Compatibility with your assay is critical. The recombinant MIP-5 you use as a standard must be recognized by the capture and detection antibodies in your ELISA. Differences in protein fragment, expression system, or post-translational modifications can affect antibody binding and assay accuracy. Some kit manuals specifically caution that standards from other sources may not be detected equivalently due to these differences.

• Purity and quantification: Ensure your recombinant MIP-5 is highly purified and accurately quantified (e.g., by BCA or Bradford assay). Impurities or inaccurate concentration assignment will compromise the reliability of your standard curve.

• Standard curve preparation: Prepare a serial dilution of your recombinant MIP-5 in the same diluent as your samples, covering the expected concentration range (e.g., 0.156–10 ng/mL as commonly used in MIP-5 ELISAs). Always run the standard curve in parallel with your samples for each assay to account for inter-assay variability.

• Validation: If you are not using the standard provided with a commercial kit, validate your recombinant MIP-5 standard by comparing its performance (e.g., parallelism, recovery) to the kit standard if possible, or by demonstrating consistent, linear standard curves and accurate quantification in spiked samples.

Limitations:

• If your recombinant MIP-5 differs in sequence, folding, or glycosylation from the native protein or the kit standard, quantification may not be directly comparable.
• Some kits explicitly state that they cannot guarantee detection of recombinant proteins from other sources, so empirical validation is necessary.

Summary:
You may use recombinant human MIP-5 as a standard for ELISA quantification if it is pure, accurately quantified, and validated for compatibility with your assay antibodies. Always prepare a fresh standard curve for each assay and validate the standard’s performance to ensure reliable results.

Recombinant Human Macrophage Inflammatory Protein-5 (MIP-5) has been validated in published research primarily for its role as a chemotactic factor and modulator of immune cell function, especially in studies involving human T-lymphocytes and monocytes.

Key validated applications include:

• Chemotaxis Assays: MIP-5 has been shown to induce significant chemotaxis of human T-lymphocytes and monocytes at concentrations ranging from 1–10 ng/mL. This is a standard assay to evaluate the ability of chemokines to attract specific immune cell populations.

• Calcium Flux Assays: MIP-5 induces calcium flux in human CCR1-transfected cells, validating its activity through receptor-mediated signaling.

• Macrophage Polarization Studies: While direct evidence for MIP-5 in macrophage polarization is limited, chemokines like MIP-5 are commonly used in research to study macrophage activation and polarization, especially in the context of inflammatory diseases.

• Disease Association Studies: Elevated circulating MIP-5 levels have been associated with increased risk of diabetic kidney disease (DKD) and renal decline in patients with type 2 diabetes mellitus, supporting its use as a biomarker in clinical research.

• Receptor Binding and Signal Transduction: MIP-5 has been validated for binding to CCR1 and CCR3 receptors, which are involved in immune cell migration and activation.

• In Vitro Functional Assays: Recombinant MIP-5 is used in vitro to study its effects on immune cell migration, activation, and signaling pathways, particularly in the context of inflammation and immune response.

Summary Table of Validated Applications

Additional Notes:

• MIP-5 is also known as CCL15 or Lkn-1.
• Most published research utilizes recombinant MIP-5 for in vitro studies; its use in in vivo models is less common but possible for mechanistic studies of chemokine function.
• The protein is not validated for clinical or diagnostic use; all applications are restricted to research contexts.

If you require protocols or more detailed mechanistic insights for any specific application, please specify the experimental context.

To reconstitute and prepare Recombinant Human Macrophage Inflammatory Protein-5 (MIP-5) for cell culture experiments, dissolve the lyophilized protein in sterile distilled water or buffer to a concentration of 0.1–1.0 mg/mL. Avoid vigorous mixing and use gentle agitation; do not vortex.

Step-by-step protocol:

1. Equilibrate materials: Allow the vial and reconstitution buffer to reach room temperature before starting.
2. Centrifuge vial: Briefly centrifuge or tap the vial to collect all lyophilized powder at the bottom.
3. Add buffer: Reconstitute in sterile distilled water or buffer (e.g., 20 mM Tris, 150 mM NaCl, pH 8.0, or 20 mM PBS, pH 7.4, 100 mM NaCl) to achieve a final concentration of 0.1–1.0 mg/mL.
4. Gentle mixing: Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation. Do not vortex or shake vigorously to avoid foaming and protein denaturation.
5. Carrier protein (optional): For long-term storage or low concentrations, add a carrier protein such as 0.1% BSA or 5% trehalose to prevent adsorption and loss of activity.
6. Aliquot and storage: After reconstitution, aliquot the solution to avoid repeated freeze/thaw cycles. Store aliquots at 4°C for short-term (2–7 days) or –20°C to –80°C for long-term.
7. Dilution for cell culture: For cell culture, further dilute the reconstituted stock in cell culture medium or appropriate buffer, ideally containing a carrier protein to minimize adsorption.

Additional notes:

• Endotoxin levels: Confirm that endotoxin levels are suitable for cell culture (<0.1 ng/μg is typical for recombinant cytokines).
• Avoid repeated freeze/thaw: This can degrade the protein and reduce biological activity.
• Use freshly prepared working solutions: For best results, prepare working dilutions immediately before use and avoid storing diluted protein for extended periods.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant MIP-5 for cell culture applications.