MIP 1a Mouse, His

What is MIP-1α Mouse, His and how is it structurally characterized?

MIP-1α Mouse, His is a recombinant version of mouse Macrophage Inflammatory Protein 1-alpha (CCL3) produced in E. coli expression systems. This protein is a single, non-glycosylated polypeptide chain containing 93 amino acids (positions 24-92 of the native sequence) with a molecular mass of approximately 10.4kDa. The recombinant protein features a 24 amino acid histidine tag at the N-terminus that facilitates purification through proprietary chromatographic techniques. The amino acid sequence is: MGSSHHHHHH SSGLVPRGSH MGSMAPYGAD TPTACCFSYS RKIPRQFIVD YFETSSLCSQ PGVIFLTKRN RQICADSKET WVQEYITDLE LNA .

What are the primary biological functions of MIP-1α in mouse models?

MIP-1α functions as a critical chemotactic cytokine (chemokine) that regulates immune cell trafficking and activation. In mouse models, MIP-1α plays several essential roles:

• Acts as a potent chemoattractant for macrophages, as demonstrated in wound healing studies where MIP-1α depletion resulted in significantly reduced macrophage infiltration (41% decrease)

• Participates in T-cell recruitment, though interestingly, MIP-1α-deficient mice show unimpaired generation of virus-specific effector T cells

• Selectively attracts CD8+ lymphocytes, distinguishing it from MIP-1β which preferentially attracts CD4+ lymphocytes

• Contributes to angiogenesis in wound healing, with neutralizing antibody studies showing diminished angiogenic response when MIP-1α is depleted

• Promotes collagen synthesis during wound repair, with hydroxyproline levels 29% lower in MIP-1α-depleted mice compared to controls

How does MIP-1α expression change during inflammatory responses?

MIP-1α expression undergoes significant temporal changes during inflammatory responses. In lysophosphatidylcholine (LPC)-induced demyelination studies, MIP-1α mRNA is typically undetectable in normal, uninjured spinal cord but increases dramatically after injury. The expression becomes detectable at 3 hours post-LPC injection and reaches peak levels between 6-24 hours, showing approximately 40-fold greater expression compared to PBS-injected controls. This temporal pattern is significant as it precedes the recruitment of monocytes, neutrophils, and T-cells (which begins around 6 hours) and activation of macrophages (12-96 hours) .

What is the optimal storage and stability protocol for MIP-1α Mouse, His?

For optimal stability and activity retention, MIP-1α Mouse, His should be stored according to these research-validated guidelines:

• For short-term use (2-4 weeks): Store at 4°C

• For long-term storage: Maintain at -20°C

• For extended storage periods: Addition of a carrier protein (0.1% HSA or BSA) is recommended

• Multiple freeze-thaw cycles should be strictly avoided to prevent protein degradation and activity loss

• Typical formulation includes 20mM Sodium citrate buffer (pH 3.5) and 10% glycerol at a concentration of 0.5mg/ml

How can researchers effectively study MIP-1α's role in T-cell-mediated immunity?

Investigating MIP-1α's role in T-cell-mediated immunity requires sophisticated experimental approaches:

• Knockout mouse studies: MIP-1α-deficient mice provide a powerful system to examine the necessity of this chemokine in T-cell responses. Research with lymphocytic choriomeningitis virus (LCMV) has demonstrated that despite the absence of MIP-1α, generation of virus-specific effector T cells proceeds normally, and T-cell-mediated virus control remains intact .

• CSF cell composition analysis: When studying neuroinflammatory responses, cerebrospinal fluid analysis is crucial. In LCMV-induced meningitis models, researchers should quantify and phenotype inflammatory cells in the CSF to assess T-cell recruitment. Notably, MIP-1α-deficient mice show no difference in cell numbers or composition of inflammatory cells in the CSF compared to wild-type mice .

• Mortality assessment: For severe inflammatory models like viral meningitis, survival analysis provides valuable insights. Both MIP-1α-deficient and wild-type mice infected intracerebrally with LCMV die around 8-10 days post-challenge, suggesting MIP-1α is not essential for the pathogenic T-cell response in this context .

• Comparison across infection models: It's critical to recognize that MIP-1α's role varies significantly between pathogens. While dispensable for LCMV T-cell responses, MIP-1α deficiency leads to increased lung viral titers in influenza infections, resistance to coxsackievirus-induced myocarditis, and reduced lung inflammation in respiratory syncytial virus infections .

What experimental considerations are important for neutralizing MIP-1α in vivo?

When designing experiments to neutralize MIP-1α in vivo, several critical factors must be addressed:

• Antibody selection: Use validated neutralizing antibodies with demonstrated specificity. Studies have successfully employed goat polyclonal antibodies (e.g., AB-450-NA, R&D Systems) .

• Delivery method: The administration route should match the target tissue. For central nervous system studies, direct microinjection into the spinal cord has been effective using 1μl cocktails containing LPC (2μg/μl) and neutralizing antibodies (0.4μg/μl) .

• Control selection: Appropriate species and isotype-specific control immunoglobulins are essential. For goat polyclonal antibodies, use goat IgG (e.g., sc-2028, Santa Cruz Biotechnology) .

• Dosing schedule: For systemic neutralization in wound healing studies, intraperitoneal injection of 0.5ml anti-murine MIP-1α antisera 2 hours before wounding and then every 2 days has proven effective .

• Confirmation of neutralization: Validate antibody presence at the target site using immunohistochemical staining for the administered immunoglobulin .

• Multi-factorial analysis: Consider neutralizing multiple chemokines/cytokines simultaneously (MCP-1, MIP-1α, GM-CSF, and TNF-α) as they often work in concert during inflammatory responses .

How can researchers accurately quantify MIP-1α in experimental samples?

Accurate quantification of MIP-1α in research samples requires validated methodologies:

Table 1: MIP-1α Quantification Methods Comparison

For ELISA-based quantification, the Mouse MIP-1α solid-phase sandwich ELISA represents the gold standard. This method employs a target-specific capture antibody pre-coated in microplate wells to which samples, standards, or controls are added. A detector antibody then completes the sandwich, followed by substrate addition that produces measurable signal proportional to MIP-1α concentration. This approach has undergone rigorous validation for criteria including sensitivity, specificity, precision, and lot-to-lot consistency .

For mRNA expression analysis, RT-PCR has been effectively used with specific primers to detect MIP-1α expression changes. This approach is particularly valuable for temporal studies examining expression kinetics following inflammatory stimuli .

How does MIP-1α contribute to wound healing processes?

MIP-1α plays multiple critical roles in wound healing through coordinated cellular recruitment and activation processes:

• Macrophage recruitment: MIP-1α serves as a potent macrophage chemoattractant in wound repair. Neutralizing antibody studies demonstrate that MIP-1α depletion leads to a significant 41% decrease in macrophage numbers at wound sites 3 days post-injury .

• Angiogenesis regulation: MIP-1α significantly influences the wound angiogenic environment. When assessed using the corneal micropocket assay for neovascularization, wound homogenates from anti-MIP-1α-treated mice show markedly diminished angiogenic responses (positive response in only 27% of corneas) compared to controls (positive in 73% of corneas) .

• Collagen synthesis: MIP-1α contributes to extracellular matrix deposition during healing. Hydroxyproline analysis of day 7 wounds reveals that MIP-1α neutralization results in a significant 29% reduction in collagen content compared to control-treated animals .

This multi-functional role highlights the importance of macrophage-derived factors in coordinating repair processes beyond simple inflammatory functions. Researchers investigating wound healing should consider these interconnected pathways when designing experiments targeting MIP-1α.

How does MIP-1α function in demyelinating disease models?

In demyelinating disease models, particularly lysophosphatidylcholine (LPC)-induced demyelination, MIP-1α exhibits distinct temporal expression patterns and functional roles:

• Rapid induction: Unlike constitutively expressed chemokines, MIP-1α mRNA is undetectable in normal mouse spinal cord but shows dramatic upregulation following LPC injection, with expression increasing at 3 hours and peaking at 6-24 hours post-injection .

• Coordinated cytokine expression: MIP-1α works in concert with other inflammatory mediators including MCP-1, GM-CSF, and TNF-α, which show varying temporal expression profiles: GM-CSF peaks at 30 minutes, TNF-α at 1 hour, and MCP-1 at 3 hours post-LPC injection .

• Immune cell recruitment mediator: The expression timing of these chemokines/cytokines precedes the recruitment of monocytes, neutrophils, and T-cells (beginning at 6 hours) and macrophage activation (12-96 hours) in the demyelinating lesions .

Researchers studying demyelinating conditions should consider these temporal relationships when designing intervention studies targeting specific phases of the inflammatory cascade.

What controls are essential when using MIP-1α Mouse, His in experimental systems?

When incorporating MIP-1α Mouse, His into research protocols, several crucial controls must be included:

• Vehicle controls: Include appropriate buffer-only conditions containing 20mM sodium citrate (pH 3.5) with 10% glycerol to control for buffer effects .

• His-tag controls: When studying protein-protein interactions or receptor binding, consider using alternative His-tagged proteins that are structurally unrelated to MIP-1α to control for non-specific His-tag effects.

• Heat-inactivated MIP-1α: Use heat-denatured MIP-1α preparations to distinguish between specific activity and non-specific protein effects.

• Species comparison controls: For cross-species studies, include both human and mouse MIP-1α preparations as both have been shown to be active on both human and murine hematopoietic cells, though potentially with different potencies .

• Related chemokine controls: Include MIP-1β (CCL4) in experimental designs, particularly when studying lymphocyte recruitment, as these chemokines have differential effects on CD8+ versus CD4+ T cells .

• Concentration gradients: Always establish full dose-response curves rather than single concentrations to determine both efficacy and potency parameters.

How does the His-tag affect experimental interpretation when using MIP-1α Mouse, His?

The 24-amino acid histidine tag present in recombinant MIP-1α Mouse, His introduces several important considerations for experimental design and interpretation:

• Protein folding and structure: While the His-tag facilitates purification, it may subtly alter protein folding or quaternary structure compared to the native protein. This is particularly relevant for chemokines whose activity depends on precise structural conformations.

• Receptor interaction dynamics: The N-terminal region of chemokines often participates in receptor binding. The presence of a His-tag in this region could potentially modify receptor interaction kinetics or binding affinity compared to native MIP-1α.

• Oligomerization potential: Chemokines including MIP-1α can form functional dimers or higher-order oligomers. The His-tag may influence these oligomerization properties, potentially altering functional characteristics in certain assays.

• Validation approaches: Researchers should consider parallel experiments with commercially available non-tagged MIP-1α or enzymatically cleaved His-tag preparations when investigating subtle functional differences.

• Buffer considerations: His-tagged proteins may exhibit different pH and salt sensitivities. The recommended storage in sodium citrate buffer (pH 3.5) should be considered when designing experiments at physiological pH .

What are the key considerations for using MIP-1α in functional migration assays?

When designing and interpreting migration assays using MIP-1α Mouse, His, researchers should address these methodological factors:

• Cell specificity: MIP-1α demonstrates differential chemotactic activity across immune cell populations. While both MIP-1α and MIP-1β attract monocytes, MIP-1α specifically recruits CD8+ T lymphocytes, while MIP-1β preferentially attracts CD4+ T lymphocytes. Assays should be designed with appropriate target cell populations .

• Concentration optimization: Chemotactic responses typically follow bell-shaped curves, with reduced migration at both very low and very high concentrations. Full dose-response experiments (typically 1-100 ng/ml range) should be performed.

• Gradient stability: Stable concentration gradients are essential for directional migration. Transwell systems should be carefully optimized for membrane pore size, chamber dimensions, and incubation times based on the specific cell type being studied.

• Positive controls: Include established chemotactic factors for the cell type under investigation (e.g., CXCL12 for lymphocytes) as positive controls.

• Receptor antagonist validation: To confirm specificity, include CCR1 and CCR5 receptor antagonists, as these represent the primary receptors for MIP-1α.

• Combined chemokine effects: In complex inflammatory environments, multiple chemokines operate simultaneously. Consider combination experiments with MCP-1, MIP-1β, or other relevant chemokines to model physiological conditions more accurately .

How should researchers interpret contradictory findings in MIP-1α knockout models?

When encountering contradictory results from MIP-1α knockout studies across different disease models, consider these interpretive frameworks:

What functional assays best demonstrate MIP-1α's role in inflammatory processes?

To comprehensively assess MIP-1α's contribution to inflammatory processes, researchers should employ multiple complementary functional assays:

Table 2: Functional Assays for Assessing MIP-1α Activity

When measuring inflammatory cell recruitment in wound models, the timing of assessment is critical. Macrophage infiltration typically peaks 3 days after injury, making this an optimal timepoint for quantitative analyses . For angiogenesis assessment, the corneal micropocket assay provides a sensitive readout of MIP-1α-dependent inflammatory signals that promote neovascularization .

For collagen synthesis evaluation, hydroxyproline quantification at day 7 post-injury represents an appropriate endpoint to capture MIP-1α's influence on tissue repair processes . These timepoints should be carefully considered when designing studies to avoid missing critical windows of MIP-1α activity.

How do MIP-1α levels correlate with disease severity across different inflammatory models?

Understanding the relationship between MIP-1α levels and disease severity requires careful consideration of several factors:

• Temporal expression profiles: In LPC-induced demyelination, MIP-1α mRNA expression increases at 3 hours post-injection and peaks at 6-24 hours, with levels approximately 40-fold greater than controls. This temporal pattern precedes cellular infiltration and may serve as an early biomarker of inflammatory intensity .

• Coordinated expression with other mediators: MIP-1α works in concert with other inflammatory factors (MCP-1, GM-CSF, TNF-α) that show distinct temporal expression profiles. The relative ratios of these factors, rather than absolute MIP-1α levels alone, may better predict disease severity .

• Cellular sources and tissue localization: Immunohistochemical analyses reveal that during inflammation, MIP-1α expression occurs predominantly in activated microglia and macrophages within lesioned tissue. The spatial distribution of expression may be more informative than total tissue levels .

• Functional consequences of neutralization: The effects of MIP-1α neutralization provide insight into its contribution to pathology. In wound models, anti-MIP-1α treatment reduces macrophage infiltration by 41%, angiogenic activity from 73% to 27% positive responses, and collagen deposition by 29% .

• Systemic versus local levels: While local tissue levels often correlate with local inflammatory activity, systemic (serum/plasma) levels measured by ELISA may reflect broader inflammatory states but with less predictive value for specific tissue pathology .

Researchers should consider these multidimensional relationships rather than simple correlations when interpreting MIP-1α measurements in disease models.

What emerging technologies show promise for studying MIP-1α biology?

Several cutting-edge approaches are transforming MIP-1α research:

• Single-cell transcriptomics: This technology enables identification of specific cellular sources of MIP-1α within heterogeneous tissues and reveals cell-specific responses to MIP-1α stimulation with unprecedented resolution.

• Optogenetic chemokine receptors: Engineering light-sensitive CCR1/CCR5 receptors allows precise temporal control over MIP-1α signaling pathways, helping delineate immediate versus delayed effects.

• Intravital multiphoton imaging: Real-time visualization of labeled immune cells responding to MIP-1α gradients in living tissues provides dynamic insights into recruitment kinetics not possible with endpoint analyses.

• CRISPR/Cas9 genome editing: Beyond conventional knockouts, precise modification of MIP-1α binding sites on receptors or targeted mutagenesis of specific protein domains enables sophisticated structure-function studies.

• Nanobody-based inhibitors: Development of single-domain antibody fragments with enhanced tissue penetration offers improved spatial control of MIP-1α neutralization compared to conventional antibodies.

These approaches address longstanding challenges in chemokine biology, particularly the redundancy and context-specificity that have complicated traditional knockout and neutralization studies.

How might targeting MIP-1α provide therapeutic opportunities in inflammatory diseases?

The research findings on MIP-1α suggest several promising therapeutic strategies:

• Wound healing applications: The critical role of MIP-1α in macrophage recruitment, angiogenesis, and collagen synthesis suggests that controlled delivery of this chemokine might enhance repair in chronic wounds characterized by insufficient macrophage activity .

• Demyelinating disease intervention: The early upregulation of MIP-1α (along with MCP-1, GM-CSF, and TNF-α) in demyelinating lesions presents a potential early intervention window. Combination therapies targeting multiple chemokines might be more effective than targeting MIP-1α alone .

• T-cell trafficking modulation: The differential effects of MIP-1α on CD8+ versus CD4+ T-cell recruitment could be exploited to selectively modulate specific T-cell subsets in autoimmune conditions .

• Virus-specific considerations: The pathogen-specific dependencies on MIP-1α highlight the importance of tailored approaches. While MIP-1α inhibition might benefit certain viral inflammatory conditions, it could potentially impair immunity to other pathogens .

• Delivery system development: Advanced delivery systems that achieve tissue-specific, temporal control over MIP-1α activity could overcome the limitations of global inhibition or supplementation approaches.

These therapeutic directions should be pursued with careful attention to the complex and context-dependent roles of MIP-1α in different disease settings.