Recombinant Human MIP-5

Recombinant Human MIP-5 (CCL15) is widely used in research applications due to its well-characterized role as a chemokine that mediates immune cell recruitment, making it a valuable tool for studying inflammation, immune responses, and related signaling pathways.

Key scientific reasons to use Recombinant Human MIP-5:

• Chemotactic Activity: MIP-5 is a potent chemoattractant for T-lymphocytes, monocytes, and eosinophils, allowing researchers to model and quantify immune cell migration in vitro and in vivo.
• Receptor Specificity: It binds to CCR1 and CCR3 chemokine receptors, enabling studies on receptor-ligand interactions, signaling cascades, and competitive binding with other chemokines such as MIP-1α and MCP-3.
• Inflammatory Disease Models: MIP-5 plays a key role in leukocyte recruitment and inflammatory disease development, making it relevant for research into autoimmune disorders, allergy, and tissue injury.
• Cellular Expression: Human MIP-5 is expressed in T and B lymphocytes, NK cells, monocytes, and dendritic cells, supporting its use in studies of immune cell function and communication.
• Functional Assays: Recombinant MIP-5 is used to induce calcium flux in CCR1-transfected cells and to measure chemotaxis, providing quantitative endpoints for cell signaling and migration assays.
• Genetic and Molecular Studies: The gene encoding MIP-5 is located in a cluster of CC chemokines on chromosome 17, facilitating genetic studies and manipulation in immunology research.

Best practices and considerations:

• For in vitro studies, both glycosylated and non-glycosylated forms are typically biologically active, but for in vivo applications, mammalian-expressed (glycosylated) recombinant proteins are preferred due to increased stability and resistance to protease degradation.
• Recombinant MIP-5 is available as a purified protein, suitable for use in cell culture, migration assays, receptor binding studies, and signaling pathway analysis.
• Always confirm the purity, activity, and source of the recombinant protein to ensure reproducibility and relevance to your experimental system.

Typical research applications include:

• Immune cell migration assays
• Receptor binding and competition studies
• Inflammatory disease modeling
• Cell signaling and calcium flux assays
• Genetic and molecular immunology research

Using recombinant human MIP-5 enables precise control over experimental conditions and reproducibility in studies of chemokine biology, immune cell trafficking, and inflammation.

You can use recombinant human MIP-5 as a standard for quantification or calibration in ELISA assays, but only under specific conditions and with important caveats. The suitability depends on the compatibility between the recombinant protein and the antibodies used in your ELISA kit, as well as the protein’s structure, post-translational modifications, and source.

Key considerations:

• Most commercial ELISA kits for MIP-5 (CCL15) use recombinant MIP-5 as their standard for generating the calibration curve. This is common practice, as recombinant proteins provide a consistent and defined standard for quantification.
• However, not all recombinant proteins are equivalent. Differences in expression systems (e.g., E. coli vs. mammalian), protein folding, glycosylation, or the presence of tags can affect antibody recognition and assay performance.
• Some ELISA kits explicitly caution against using recombinant proteins from sources other than the kit’s own standard, due to potential differences in epitope presentation or structure. For example:

  “The standard of the kit and immunogen used for antibody preparation are commonly recombinant proteins, as different fragments, expression systems, purification methods might be used in recombinant protein preparation, we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to use the kit for the detection of recombinant protein.”

• Some kits are designed to detect only native MIP-5, not recombinant forms. Always check your kit’s documentation for such restrictions.

Best practices:

• If your ELISA kit provides its own recombinant MIP-5 standard, use that for calibration. This ensures compatibility with the capture and detection antibodies.
• If you wish to use a recombinant MIP-5 from another source as a standard, you must validate that it is recognized equivalently by the kit’s antibodies. This typically involves running a parallel standard curve with both the kit standard and your recombinant protein to compare their performance.
• Be aware of potential quantification discrepancies due to differences in protein structure, purity, or concentration determination methods.

Summary Table: Recombinant MIP-5 as ELISA Standard

In conclusion:
You can use recombinant human MIP-5 as a standard for ELISA quantification if it is validated to be recognized by your assay’s antibodies and is structurally similar to the native protein or the kit’s standard. Always consult your kit’s manual and, if in doubt, perform a side-by-side comparison to ensure accurate quantification.

Recombinant Human MIP-5 (also known as CCL15) has been validated primarily for use in functional bioassays measuring chemotactic activity, especially for its ability to attract human T-lymphocytes and monocytes in vitro. The most widely documented application is the assessment of chemotaxis, where MIP-5 induces migration of T cells at concentrations as low as 1–10 ng/mL.

Additional validated applications in published research include:

• Bioactivity assays: Used to confirm the protein’s ability to induce chemotaxis of T-lymphocytes, a hallmark of its function as a chemokine.
• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant MIP-5 is used as a standard or control in ELISA kits designed to quantify MIP-5 in biological samples.
• Specificity testing in immunoassays: Recombinant MIP-5 is used to assess the specificity of antibodies or detection reagents in multiplex immunoassays.
• Receptor binding studies: While not as commonly cited as chemotaxis, recombinant MIP-5 can be used to study interactions with its primary receptor, CCR1, in cell-based or biochemical assays.

Key details:

• Chemotaxis assays are the most established and validated application, with published protocols specifying the use of recombinant MIP-5 to induce migration of T cells but not monocytes or granulocytes.
• ELISA validation: Recombinant MIP-5 is used as a calibrator or positive control in commercial and custom ELISA kits for human samples.
• Immunoassay specificity: Used to confirm the selectivity of detection reagents in multiplexed cytokine panels.

There is no evidence in the provided search results that recombinant human MIP-5 has been validated for use in Western blotting, immunohistochemistry, or in vivo animal models as a functional reagent. Its primary use remains in in vitro functional and immunoassay applications.

Summary of validated applications:

• Chemotaxis/functional bioassays (T-lymphocyte migration)
• ELISA (as standard/control)
• Immunoassay specificity testing
• Receptor binding/interaction studies

If you require protocols or further technical details for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Human MIP-5 (CCL15) protein for cell culture experiments, follow these best-practice steps:

1. Equilibrate the lyophilized vial and your chosen reconstitution buffer (typically sterile, distilled water or a mild buffer) to room temperature before opening.

2. Centrifuge or tap the vial briefly to ensure all lyophilized material is at the bottom.

3. Add sterile water or buffer:

   • Use sterile, distilled water to achieve a concentration of 0.1–1.0 mg/mL.
   • Alternatively, some protocols recommend using 20 mM Tris, 150 mM NaCl, pH 8.0 for reconstitution.
   • The minimum recommended concentration is ≥100 μg/mL.

4. Gently mix:

   • Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation (do not vortex or shake vigorously, as this can denature the protein).
   • If undissolved particles remain, gently pipette up and down or let stand longer; do not vortex.

5. Dilution for cell culture:

   • After initial reconstitution, dilute the protein to your working concentration using aqueous buffer containing a carrier protein (e.g., 0.1–1% BSA or HSA) to minimize adsorption and stabilize the protein.
   • Prepare all further dilutions in cell culture medium or buffer containing carrier protein.

6. Aliquot and storage:

   • Use the reconstituted protein immediately or aliquot into single-use portions to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C or –80°C for long-term stability.
   • For short-term use (up to one week), storage at 2–8°C is acceptable.

Additional notes:

• Avoid dissolving the protein at elevated temperatures (do not use 37°C).
• Always use sterile technique to prevent contamination.
• If using for cell culture, ensure all buffers and solutions are endotoxin-free.

Summary Table: Recombinant Human MIP-5 Reconstitution

These steps will help preserve the biological activity and integrity of recombinant MIP-5 for reliable cell culture experiments.