Recombinant Human MIP-1β

Recombinant Human MIP-1β (also known as CCL4) is widely used in research due to its critical roles in immune cell recruitment, inflammation, and disease modeling, particularly in studies involving chemokine signaling, immune cell function, and HIV research.

Key reasons to use Recombinant Human MIP-1β in research applications:

• Chemotaxis and Immune Cell Recruitment: MIP-1β is a potent chemoattractant for various immune cells, including monocytes, CD4+ T lymphocytes, B cells, eosinophils, and dendritic cells. It is especially notable for selectively attracting CD4+ lymphocytes, distinguishing its function from MIP-1α, which attracts CD8+ lymphocytes.

• Inflammatory Response Modeling: MIP-1β is a proinflammatory cytokine involved in both acute and chronic inflammatory responses, making it valuable for studying mechanisms of inflammation and immune regulation. It activates granulocytes (neutrophils, eosinophils, basophils), leading to acute neutrophilic inflammation.

• Functional Studies of Chemokine Receptors: MIP-1β binds with high affinity to the CCR5 receptor. This interaction is crucial for research on chemokine receptor signaling, immune cell migration, and receptor desensitization.

• HIV Research: MIP-1β is significant in HIV pathogenesis studies because it can inhibit the entry of M-tropic HIV-1 into T cells by binding to and downregulating CCR5, a coreceptor for HIV-1. This property is exploited in studies of viral infection, immune evasion, and therapeutic intervention.

• Tumor Immunology and Cytotoxicity: MIP-1β can augment the cytotoxic activity of peripheral blood monocytes (PBM) and enhance the release of inflammatory cytokines such as IL-1 and TNF-α, particularly in healthy individuals. This makes it useful for modeling tumor-immune interactions and studying monocyte-mediated tumor cell killing.

• Bone Biology and Disease Models: Both MIP-1α and MIP-1β play roles in bone resorption and osteolytic lesion development, relevant for research in multiple myeloma and bone metastasis.

• Standardization and Reproducibility: Recombinant proteins provide high purity, defined activity, and batch-to-batch consistency, which are essential for reproducible experimental results.

• Versatility in Assays: Recombinant MIP-1β is suitable for a range of applications, including bioassays, chemotaxis assays, ELISA standards, and cell signaling studies.

In summary, Recombinant Human MIP-1β is a versatile tool for dissecting immune mechanisms, modeling disease processes, and developing therapeutic strategies, especially where chemokine signaling and immune cell migration are central to the research question.

Yes, recombinant human MIP-1β 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 ELISA protocols for cytokines and chemokines such as MIP-1β (CCL4).

Supporting details:

• Standard Curve Preparation: Quantitative ELISA assays require a standard curve generated from known concentrations of the target protein. Recombinant human MIP-1β, when properly quantified and diluted, serves as an appropriate standard for this purpose.
• Assay Validation: Commercial ELISA kits for human MIP-1β routinely use recombinant protein as the calibration standard. These kits demonstrate that recombinant MIP-1β yields parallel standard curves to those generated with natural protein, indicating suitability for quantification.
• Purity and Quantification: The recombinant protein should be highly purified and its concentration verified (e.g., by HPLC or absorbance at 280 nm) to ensure accurate calibration.
• Matrix Compatibility: Recovery experiments show that recombinant MIP-1β standards perform reliably across various sample matrices (serum, plasma, cell culture supernatant), with recoveries typically near 100%.
• Protocol Considerations: Follow standard ELISA protocols for dilution and handling of the recombinant standard. Most protocols recommend preparing a serial dilution covering the expected range of sample concentrations (e.g., 0–1000 pg/mL).

Additional notes:

• Bioassay Use: While recombinant MIP-1β is suitable for ELISA calibration, it may not be validated for functional bioassays unless specifically tested for biological activity.
• Lot-to-Lot Consistency: For reproducibility, ensure that the recombinant standard is consistent between lots, or normalize to a reference batch if possible.
• Documentation: Always consult the datasheet or certificate of analysis for your recombinant protein to confirm its suitability for use as an ELISA standard.

In summary, recombinant human MIP-1β is widely accepted and validated as a standard for quantitative ELISA assays, provided best practices for protein handling and assay setup are followed.

Recombinant Human MIP-1β (CCL4) has been validated for a range of applications in published research, primarily in immunology, cell signaling, and disease models. Key validated applications include:

• Chemotaxis assays: MIP-1β is widely used to assess chemotactic responses of various immune cells, including monocytes, CD4+ lymphocytes, B cells, eosinophils, and dendritic cells. Its ability to chemoattract cells expressing CCR5 is a standard functional validation.

• Cytokine release and activation assays: MIP-1β has been used to stimulate peripheral blood monocytes (PBM) to induce cytokine release (e.g., IL-1) and to generate tumoricidal monocytes, with differential effects observed in healthy donors versus cancer patients.

• Receptor binding and signaling studies: Recombinant MIP-1β is validated in competitive binding assays (e.g., displacement of radiolabeled chemokines), GTPγS exchange assays, intracellular calcium flux, and chemotaxis assays to characterize its interaction with chemokine receptors such as CCR1 and CCR5.

• Bioassays for functional activity: Its biological activity is routinely validated by its ability to induce chemotaxis in cell lines transfected with human CCR5 (e.g., BaF/3 cells).

• ELISA and immunoassays: MIP-1β is used as a standard or calibrator in ELISA-based quantification of chemokines in plasma or cell culture supernatants, including in clinical biomarker studies.

• HIV research: MIP-1β is validated as a natural inhibitor of HIV-1 entry by binding to CCR5, and is used in assays studying HIV-1 infection and chemokine-mediated inhibition.

• Disease models: It is used in studies of inflammatory diseases (e.g., arthritis, multiple sclerosis), cancer (e.g., breast cancer, multiple myeloma), and bone metabolism (osteolytic lesion development).

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data from multiple sources. If you need protocols or more technical details for a specific application, please specify the assay or research context.

To reconstitute Recombinant Human MIP-1β (CCL4) for cell culture experiments, dissolve the lyophilized protein in sterile buffer—typically sterile PBS or distilled water—to a concentration of 100 μg/mL, and include at least 0.1% serum albumin (human or bovine) if recommended to stabilize the protein and prevent adsorption to surfaces. Avoid vigorous mixing and foaming during reconstitution.

Detailed protocol:

• Preparation:

  • Equilibrate the vial to room temperature before opening to minimize condensation.
  • Use aseptic technique throughout to prevent contamination.

• Reconstitution:

  • Add sterile PBS (phosphate-buffered saline) to the vial. For most applications, reconstitute at 100 μg/mL.
  • If the formulation contains acetonitrile and TFA, ensure complete dissolution by gently swirling or tapping the vial. Do not vortex or shake vigorously to avoid foaming.
  • If recommended, add 0.1% serum albumin (BSA or HSA) to the buffer to stabilize the protein, especially for low concentration working solutions.
  • Allow the protein to dissolve for 15–30 minutes at room temperature. Inspect visually to confirm complete dissolution.

• Aliquoting and Storage:

  • Once dissolved, aliquot the solution into sterile microcentrifuge tubes to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C to –70°C for long-term storage. After reconstitution, the protein is stable for up to 1 month at 2–8°C or 3 months at –20°C to –70°C under sterile conditions.
  • Avoid repeated freeze-thaw cycles, which can degrade the protein.

• Working Solution Preparation:

  • For cell culture, dilute the stock solution to the desired working concentration using the same medium used for your cells (e.g., RPMI, DMEM, etc.), ideally supplemented with 0.1% albumin to maintain stability.
  • Prepare fresh working solutions before each experiment.

Best Practices:

• Always check the product-specific datasheet for any unique requirements regarding buffer composition or reconstitution procedure.
• Avoid prolonged exposure to room temperature and light.
• Use polypropylene tubes for dilutions to minimize protein adsorption.
• Mix gently and avoid foaming, which can denature the protein.

Summary Table:

This protocol ensures optimal stability and biological activity of recombinant MIP-1β for cell culture applications.