Recombinant Human MIP-1α

Recombinant Human MIP-1α (Macrophage Inflammatory Protein-1 alpha, also known as CCL3) is widely used in research to study immune cell recruitment, inflammation, tissue repair, and antiviral responses. Its use enables controlled, reproducible experiments on the biological functions and signaling pathways of this chemokine.

Key scientific reasons to use recombinant human MIP-1α in research applications:

• Immune Cell Recruitment and Activation: MIP-1α is a potent chemoattractant for monocytes, macrophages, and other immune cells, playing a central role in orchestrating inflammatory responses. Using the recombinant protein allows you to directly assess its effects on cell migration, activation, and cytokine production in vitro or in vivo.

• Inflammation and Disease Modeling: MIP-1α is implicated in acute and chronic inflammatory diseases, including brain injury, cancer, and autoimmune disorders. Recombinant MIP-1α can be used to model these conditions, dissect signaling pathways, and evaluate anti-inflammatory interventions.

• Wound Healing and Tissue Repair: MIP-1α promotes wound healing by recruiting macrophages to sites of tissue injury, making it valuable for studies on tissue regeneration and repair mechanisms.

• Antiviral and Antitumor Research: MIP-1α, alone or as a heterodimer with MIP-1β, exhibits direct antiviral activity (notably against HIV-1 and HSV-1) and enhances tumoricidal activity of monocytes, supporting its use in studies of host defense and immunotherapy.

• Receptor and Signaling Studies: MIP-1α binds to chemokine receptors CCR1, CCR4, and CCR5, which are important in immune cell signaling and are also HIV co-receptors. Recombinant protein is essential for receptor binding assays, signaling pathway analysis, and drug screening.

• Standardization and Reproducibility: Recombinant proteins provide a consistent, defined reagent, critical for quantitative assays (e.g., ELISA standards, bioassays) and for comparing results across experiments and laboratories.

• Species-Specific Activity: Using human recombinant MIP-1α ensures relevance for studies involving human cells, tissues, or translational research, as chemokine activity can be species-specific.

Common applications include:

• Chemotaxis and migration assays
• Cytokine release and immune activation studies
• In vitro and in vivo inflammation models
• Antiviral and antitumor functional assays
• Receptor binding and signaling pathway analysis

In summary, recombinant human MIP-1α is a versatile tool for dissecting immune mechanisms, modeling disease, and developing therapeutic strategies targeting inflammation, infection, and cancer.

Yes, you can use recombinant human MIP-1α as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately known.

Supporting details and best practices:

• Recombinant proteins are commonly used as ELISA standards because they provide a defined, consistent source of the analyte, allowing for the generation of a standard curve against which unknown samples can be quantified.
• Many commercial ELISA kits for human MIP-1α (also known as CCL3) use recombinant human MIP-1α as the standard, and their protocols demonstrate that results for natural and recombinant MIP-1α are comparable and yield parallel standard curves.
• When preparing your standard curve, ensure the recombinant protein is reconstituted and diluted according to best practices to maintain accuracy and reproducibility.
• The standard curve should cover the expected concentration range of your samples (commonly 0–1000 pg/mL, but may extend up to 3000 pg/mL depending on assay sensitivity and sample type).
• Confirm that your ELISA antibodies recognize both natural and recombinant forms of MIP-1α, as most commercial kits are validated for both.
• Use only carrier-free, highly purified recombinant protein for standard preparation to avoid interference from stabilizers or other proteins.

Limitations and considerations:

• The recombinant standard should match the isoform and sequence of the endogenous MIP-1α you expect in your samples, as there are closely related isoforms (e.g., CCL3/LD78a and CCL3L1/LD78b) with high homology but potentially different immunoreactivity.
• Always verify that your recombinant standard is compatible with your specific ELISA kit or antibody pair, as some kits may have specific requirements or recommendations regarding the standard used.
• For highest accuracy, run a standard curve with each assay and do not extrapolate beyond the highest standard point.

In summary, recombinant human MIP-1α is suitable and widely used as a standard for ELISA quantification, provided it is properly prepared and validated for your assay system.

Recombinant Human MIP-1α (CCL3) has been validated for several key applications in published research, primarily in bioassays, binding assays, and clinical biomarker quantitation.

Validated Applications:

• Bioassays:
  Used extensively to study chemotactic activity, immune cell recruitment (e.g., granulocytes, monocytes, lymphocytes, eosinophils, dendritic cells), and cytokine signaling in various cell types. These bioassays have been applied in research on HIV infection, immune modulation, inflammatory responses, and bone resorption in multiple myeloma.

• Binding Assays:
  Radiolabeled recombinant MIP-1α (e.g., I-125) is used as a tracer in receptor binding assays to characterize interactions with chemokine receptors (CCR1, CCR4, CCR5), determine receptor expression levels, and study ligand-receptor kinetics (e.g., dissociation constants, inhibitor constants).

• Multiplex Immunoassays & ELISA Standards:
  Validated for use as a standard in ELISA and multiplex immunoassays to quantify MIP-1α levels in human serum and other biological fluids. These assays are used in clinical studies as pharmacodynamic and safety biomarkers, particularly in contexts such as HIV infection, allergic asthma, sepsis, and cancer.

• Functional Assays:
  Demonstrated activity in assays measuring chemotaxis, cytokine secretion, and synergistic effects with other chemokines and cytokines (e.g., IFN-γ, MIP-1β, RANTES) in driving Th1 immune responses and upregulating inflammatory markers (e.g., CD40, TNF-α, IL-12).

• Cell Culture Studies:
  Used to stimulate or modulate immune cells in vitro, including studies on phagocytosis, integrin regulation, and chemokine secretion in primary human cells.

Representative Published Research Applications:

• HIV Vaccine and Infection Studies:
  Investigating Fc receptor-mediated phagocytosis and chemokine cooperation in monocyte and lymphocyte migration.

• Inflammatory and Autoimmune Disease Models:
  Studying chemokine secretion, integrin regulation, and immune cell recruitment in diseases such as allergic asthma, sepsis, and chronic prostatitis.

• Cancer Research:
  Assessing MIP-1α as a biomarker for bone resorption and lytic bone lesions in multiple myeloma.

• Immunology:
  Exploring the role of MIP-1α in Th1/Th2 immune responses, cytokine synergy, and macrophage activation.

Summary Table of Validated Applications

Key Notes:

• MIP-1α is validated for use in both in vitro and in vivo studies.
• It is a standard tool for immunology, inflammation, infectious disease, and oncology research.
• Validation includes precision, accuracy, sensitivity, and stability for clinical biomarker assays.

If you require protocol details or specific assay recommendations, please specify the intended application.

To reconstitute and prepare Recombinant Human MIP-1α (CCL3) for cell culture experiments, follow these best-practice steps:

1. Equilibrate and Centrifuge:

   • Allow the lyophilized vial to reach room temperature before opening to minimize condensation.
   • Briefly centrifuge the vial to ensure all powder is at the bottom.

2. Reconstitution Buffer:

   • Use sterile water or sterile PBS for reconstitution. If the protein is supplied without carrier protein, add at least 0.1% BSA (bovine serum albumin) or 0.1% HSA (human serum albumin) to the buffer to stabilize the protein and prevent adsorption to plastic.
   • Typical reconstitution concentrations are 0.1–1.0 mg/mL (for example, add 100 μL–1 mL buffer per 100 μg protein).

3. Dissolving the Protein:

   • Gently add the buffer down the side of the vial. Do not vortex; instead, gently pipette up and down or swirl to dissolve.
   • Allow the solution to sit at room temperature for 10–30 minutes to ensure complete dissolution. If undissolved material remains, gently mix for up to 2 hours.

4. Aliquoting and Storage:

   • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C to –80°C for long-term storage. For short-term use (up to 1 month), store at 2–8°C.
   • Avoid repeated freeze-thaw cycles, as this can degrade the protein.

5. Preparation for Cell Culture:

   • Before use, dilute the reconstituted stock to the desired working concentration in cell culture medium or assay buffer, ideally containing 0.1% BSA to maintain stability.
   • Filter sterilize if necessary, using a 0.2 μm filter, especially if sterility is critical for your application.

Summary Table: Recombinant Human MIP-1α Reconstitution

Key technical notes:

• Always consult the specific product datasheet for any manufacturer-specific recommendations, as formulation and stabilizers may vary.
• For bioassays, typical working concentrations range from 1–100 ng/mL, but optimal dosing should be empirically determined for your cell type and assay.
• If the protein is used for sensitive applications, consider filter sterilization after reconstitution.

These steps will help ensure protein stability, activity, and reproducibility in your cell culture experiments.