MIP 4 Human, His

What is MIP-4/CCL18 Human, His and what are its fundamental structural characteristics?

MIP-4/CCL18 Human recombinant protein with histidine tag is a single, non-glycosylated polypeptide chain containing 93 amino acids, consisting of the MIP-4 sequence (typically spanning positions 22-89 of the native protein) with an additional 25 amino acid His-tag at the N-terminus. It has a molecular mass of approximately 10.4-13 kDa and is produced in E. coli expression systems . The protein is typically purified to greater than 95% purity as determined by SDS-PAGE .

The amino acid sequence is: "MGSSHHHHHH SSGLVPRGSH MGSHMQVGTN KELCCLVYTS WQIPQKFIVD YSETSPQCPK PGVILLTKRG RQICADPNKK WVQKYISDLK LNA" . This chemokine belongs to the CC chemokine family and plays significant roles in immune cell regulation and inflammatory processes.

What receptors does MIP-4/CCL18 interact with and what are their biological implications?

MIP-4/CCL18 binds to multiple receptors that mediate its diverse biological functions:

Research has specifically identified CCR8 as an important receptor for MIP-4/CCL18 . Through these receptor interactions, MIP-4/CCL18 plays roles in inflammation, cancer progression, and immune cell migration. In breast cancer models, MIP-4/CCL18 from tumor-associated macrophages promotes angiogenesis . In bladder cancer, it enhances migration, invasion, and epithelial-mesenchymal transition (EMT) by binding CCR8 .

How is recombinant MIP-4 Human, His typically produced and what purification methods are used?

Recombinant MIP-4/CCL18 Human with His-tag is produced through a standardized biotechnological process:

• Expression System: E. coli is the predominant expression system used for production

• Genetic Construction: The gene sequence coding for amino acids A21-A89 of human MIP-4/CCL18 is cloned into an expression vector with an N-terminal His-tag (typically containing 6 histidine residues)

• Purification: Following bacterial expression, the protein undergoes proprietary chromatographic techniques, with immobilized metal affinity chromatography (IMAC) being the principal method leveraging the His-tag's affinity for nickel or cobalt ions

• Quality Control: The final product is validated for purity (>95%) using SDS-PAGE analysis

• Formulation: The purified protein is typically formulated in 10mM sodium citrate buffer (pH 3.5) containing 10% glycerol at concentrations around 0.25 mg/ml

This standardized production method ensures high-quality protein suitable for research applications while maintaining consistency between batches.

What are optimal storage and working conditions for MIP-4/CCL18 experiments?

For optimal experimental results with MIP-4/CCL18 Human, His, researchers should follow these storage and handling guidelines:

For functional assays, researchers should note that the pH of the working solution should be physiologically relevant (pH 7.2-7.4), which may require buffer exchange or dilution of the storage buffer. Additionally, calcium and magnesium ions (1-2 mM) are often beneficial for chemokine receptor interactions.

How can researchers verify the biological activity of recombinant MIP-4 Human, His?

Verifying the biological activity of recombinant MIP-4/CCL18 requires multiple complementary approaches:

• Chemotaxis Assays: MIP-4/CCL18 is chemotactic for T lymphocytes but not monocytes . Transwell migration assays using primary T cells or CCR8-expressing cell lines can confirm functional activity.

• Receptor Binding Studies: Direct binding assays using surface plasmon resonance (SPR) or radioligand competition assays with cells expressing CCR8, PITPNM3, or GPR30 can verify receptor-ligand interactions.

• Signaling Activation Assays: Measuring downstream signaling events in receptor-expressing cells:

  • Calcium flux assays using fluorescent indicators

  • Phosphorylation of ERK1/2, Akt, or other signaling molecules

  • β-arrestin recruitment assays for receptor activation

• CCR3 Antagonism Verification: Competitive binding assays with CCR3 agonists (eotaxins) can confirm MIP-4's antagonistic activity on this receptor.

All activity assays should include appropriate controls:

• Positive control: Native (non-His-tagged) MIP-4/CCL18 when available

• Negative controls: Heat-denatured protein, buffer-only treatments

• Specificity controls: Irrelevant His-tagged proteins of similar size

What are recommended experimental models for studying MIP-4/CCL18's roles in inflammation and cancer?

For comprehensive investigation of MIP-4/CCL18's biological roles, researchers should consider multiple experimental models:

When selecting models, researchers should verify expression of relevant receptors (CCR8, PITPNM3, GPR30) in the experimental system. Studies indicate that MIP-4/CCL18 specifically induces chemotaxis in T lymphocytes but not monocytes, making T cells a critical model for chemotactic studies .

What techniques are most effective for studying MIP-4/CCL18 interactions with its receptors?

For detailed analysis of MIP-4/CCL18 receptor interactions, researchers should employ these complementary techniques:

• Surface-Based Real-Time Methods:

  • Surface Plasmon Resonance (SPR): Provides kinetic parameters (kon, koff, KD) for purified receptor-ligand interactions

  • Bio-Layer Interferometry (BLI): Alternative to SPR with lower sample consumption

• Solution-Based Methods:

  • Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters (ΔH, ΔS, ΔG)

  • Microscale Thermophoresis (MST): Requires minimal protein amounts

• Cell-Based Approaches:

  • Radioligand binding assays using 125I-labeled MIP-4/CCL18

  • Flow cytometry with fluorescently-labeled chemokine

  • FRET/BRET-based proximity assays for receptor interaction in living cells

• Molecular Methods:

  • Cross-linking studies followed by mass spectrometry to identify binding sites

  • Mutagenesis studies to map critical residues for interaction

When examining MIP-4/CCL18 interaction with transmembrane receptors like CCR8, considerations for receptor presentation (detergent-solubilized vs. membrane preparations) are crucial for obtaining physiologically relevant data.

How should researchers design controls when using MIP-4 Human, His in cell-based assays?

Robust control design is critical for reliable MIP-4/CCL18 experimental results:

All experiments should include technical replicates (minimum triplicates) and be repeated at least three times independently to ensure reproducibility. For genetic manipulation experiments (siRNA, CRISPR), appropriate non-targeting controls must be included.

How can researchers differentiate between specific and non-specific effects of MIP-4/CCL18?

Distinguishing specific from non-specific effects of MIP-4/CCL18 requires a systematic approach:

• Receptor Dependency Testing:

  • Compare effects in receptor-positive vs. receptor-negative cells

  • Use receptor knockdown/knockout approaches (siRNA, CRISPR-Cas9)

  • Apply receptor-blocking antibodies or small molecule inhibitors

• Pharmacological Profiling:

  • Establish concentration-response relationships (specific effects typically show saturation)

  • Compare effects with structurally related chemokines

  • Competitive displacement with receptor-specific ligands

• Signaling Pathway Analysis:

  • Verify activation of canonical chemokine signaling pathways

  • Use pathway-specific inhibitors to confirm mechanism

  • Compare temporal profiles (specific effects usually occur rapidly)

• Protein Quality Controls:

  • Compare effects of His-tagged vs. non-tagged MIP-4/CCL18

  • Use heat-denatured protein as negative control

  • Employ size-fractionated samples to eliminate aggregate effects

• Gain-of-Function Approaches:

  • Express receptors in non-responsive cells to confer responsiveness

  • Reconstitute signaling in receptor-knockout models

The combination of these approaches provides strong evidence for specific receptor-mediated effects versus non-specific protein interactions.

What are the methodological considerations for studying MIP-4/CCL18-induced immune cell migration?

For rigorous assessment of MIP-4/CCL18's effects on immune cell migration, researchers should consider:

• Assay Selection and Design:

  • Transwell (Boyden chamber) assays: Standard method for quantifying directed migration

  • Microfluidic devices: Enable precise gradient formation and real-time visualization

  • 3D migration systems: Better reflect in vivo extracellular matrix environments

  • Live-cell imaging: Allows detailed analysis of migration parameters (velocity, directionality)

• Critical Parameters:

  • Gradient stability: Verify maintenance of gradients throughout experiment

  • Cell density: Optimize to avoid contact inhibition or clustering effects

  • Membrane properties: Pore size selection based on cell type (typically 5-8 μm for lymphocytes)

  • Incubation time: Optimize for cell type (2-4 hours for lymphocytes)

• Analytical Approaches:

  • Checkerboard analysis: Distinguishes chemotaxis from chemokinesis

  • Single-cell tracking: Provides detailed migration parameters

  • Endpoint quantification: Flow cytometry or fluorescence microscopy

• Experimental Controls:

  • Positive control: Known chemoattractants (e.g., CXCL12 for T cells)

  • Concentration range: Typically 1-100 ng/mL, with full dose-response curves

  • Receptor blocking: Antibodies against CCR8 or other relevant receptors

  • Signaling inhibitors: PI3K, MAPK, or Rho GTPase inhibitors to identify pathways

Research indicates that MIP-4/CCL18 specifically induces chemotaxis in T lymphocytes but not monocytes, making proper cell selection crucial for migration studies .

How does MIP-4/CCL18 contribute to cancer progression and what research models best capture this?

MIP-4/CCL18's role in cancer involves multiple mechanisms that researchers can investigate:

• Cancer-Related Functions:

  • Promotion of angiogenesis in breast cancer through tumor-associated macrophages

  • Enhancement of migration, invasion, and epithelial-mesenchymal transition (EMT) in bladder cancer via CCR8 binding

  • Potential modulation of tumor microenvironment immune responses

• Recommended Research Models:

• Methodological Approaches:

  • Analysis of CCL18 expression in patient samples correlated with clinical outcomes

  • Neutralization studies using anti-CCL18 antibodies in cancer models

  • Receptor signaling investigation in cancer cell lines expressing CCR8, PITPNM3, or GPR30

  • Selective inhibition of MIP-4/CCL18 production in tumor-associated macrophages

Future research should focus on the translational potential of targeting the MIP-4/CCL18 axis in cancer therapy, particularly in breast and bladder cancers where specific effects have been documented .

What is the relationship between MIP-4/CCL18 and other chemokines in inflammatory processes?

Understanding MIP-4/CCL18's position in the broader chemokine network requires sophisticated experimental approaches:

• Comparative Analysis:

  • Receptor sharing and competition: MIP-4/CCL18 interactions with CCR8 may compete with other CCR8 ligands

  • Signaling pathway overlap: Compare phosphorylation patterns with other chemokines

  • Functional redundancy: Assess compensatory mechanisms in knockout models

• Co-expression Studies:

  • Analyze MIP-4/CCL18 co-expression with other chemokines in disease tissues

  • Investigate coordinated regulation in response to inflammatory stimuli

  • Examine sequential chemokine expression in disease progression

• Functional Interplay:

  • Synergy/antagonism assays combining MIP-4/CCL18 with other chemokines

  • Heterologous desensitization: Pre-treatment with one chemokine affecting responses to others

  • Receptor modulation: How MIP-4/CCL18 affects expression of other chemokine receptors

• Systems Biology Approaches:

  • Chemokine network modeling in specific disease contexts

  • Multi-parameter analysis of chemokine signatures in patient samples

  • CRISPR screens to identify regulatory nodes in chemokine expression

Research should consider MIP-4/CCL18's unique position as both a CCR8 agonist and CCR3 antagonist , suggesting a complex regulatory role in balancing different aspects of immune cell function.