Recombinant Mouse C-C motif chemokine 4 protein (Ccl4) (Active)

What is Recombinant Mouse C-C Motif Chemokine 4 (CCL4) protein?

Recombinant Mouse CCL4, also known as Macrophage inflammatory protein-1beta (MIP-1beta), is a 7.8 kDa cytokine comprising 69 amino acid residues. It belongs to the C-C chemokine family and is primarily secreted by neutrophils, monocytes, B cells, T cells, fibroblasts, endothelial cells, and epithelial cells. CCL4 plays crucial roles in immune responses, particularly in the recruitment of immune cells including lymphocytes, monocytes, and leukocytes. It also participates in responses to IL-1 and IFNgamma, and contributes to TNF production when binding to its receptor CCR5 .

What are the molecular characteristics of Recombinant Mouse CCL4?

The molecular characteristics of Recombinant Mouse CCL4 include:

• UniProt Accession: P14097

• Molecular Weight: 7.8 kDa (tag-free form) to 11.8 kDa (His-tagged form)

• Amino Acid Sequence: APMGSDPPTSCCFSYTSRQLHRSFVMDYYETSSLCSKPAVVFLTKRGRQICANPSEPWVTEYMSDLELN

• Expression Range: 24-92aa (full length of mature protein)

• Structure: Contains characteristic C-C chemokine fold with conserved cysteine residues

The tag-free version of the protein is typically preferred for functional studies as it more closely resembles the native form found in vivo, while His-tagged versions may be advantageous for purification and certain binding studies .

How is the biological activity of Recombinant Mouse CCL4 assessed?

The biological activity of Recombinant Mouse CCL4 is primarily determined through chemotaxis bioassays using human monocytes. Active CCL4 typically exhibits chemotactic activity in a concentration range of 20-100 ng/ml. The protein's activity can also be assessed through its ability to bind its receptor CCR5 and initiate downstream signaling cascades. In experimental settings, functional CCL4 should demonstrate greater than 97% purity as determined by SDS-PAGE and HPLC analysis, with endotoxin levels below 1.0 EU/μg as measured by the LAL method to ensure experimental results are not confounded by endotoxin-mediated effects .

What are the optimal storage and reconstitution protocols for Recombinant Mouse CCL4?

For optimal maintenance of Recombinant Mouse CCL4 activity:

Storage protocol:

• Store lyophilized protein at -20°C/-80°C upon receipt for up to 1 year

• For reconstituted protein, aliquot to avoid repeated freeze-thaw cycles

• Store working aliquots at -20°C/-80°C for long-term storage, or at 4°C for short-term use (up to one week)

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (commonly 50%) for long-term storage

• For lyophilized preparations, the buffer before lyophilization typically consists of Tris/PBS-based buffer with 6% Trehalose, pH 8.0

These storage and reconstitution methods help maintain protein stability and biological activity over time .

How can Recombinant Mouse CCL4 be used in neuroprotection studies?

Based on research findings, Recombinant Mouse CCL4 can be used in neuroprotection studies following this methodological approach:

• Preparation of neuronal cultures: Culture primary cerebellar granule cells (neurons) at a density of 1 × 10^6 cells/mL in appropriate medium (e.g., Neurobasal A containing 2% B25, 1% FBS, and 25 mM KCl) in multi-well plates for approximately 2 weeks.

• Pretreatment with CCL4: Add recombinant CCL4 at varying concentrations to the culture medium 1 hour before exposure to neurotoxic agents.

• Neurotoxic challenge: Expose the neuronal cultures to neurotoxic compounds (such as 5 μM methylmercuric chloride [MeHg]) for 24 hours.

• Viability assessment: Measure cell viability using appropriate assays such as the alamarBlue® assay, which has been shown to effectively quantify the protective effects of CCL4.

Studies have demonstrated that recombinant CCL4 exhibits dose-dependent protective effects against MeHg-induced neurotoxicity, with significant protection observed at concentrations as low as 10 ng/mL .

What methods can be used to study CCL4 expression in response to cellular stress?

To study CCL4 expression in response to cellular stress, researchers can employ the following methodological approach:

• Cell culture system selection: Choose appropriate cell lines or primary cultures that express CCL4, such as C17.2 neural stem cells or primary neural cultures.

• Stress induction: Expose cells to stressors of interest (e.g., 10 μM methylmercuric chloride) for various time periods to establish a temporal profile of response.

• RNA extraction and quantification: Extract total RNA from cells at different time points following exposure to stressors.

• Quantitative RT-PCR analysis: Measure CCL4 mRNA levels using quantitative real-time PCR with appropriate primers specific for mouse CCL4.

• Protein analysis: Confirm changes in CCL4 protein expression using techniques such as ELISA, Western blotting, or immunocytochemistry.

• Correlation with cellular outcomes: Simultaneously assess cell viability (using assays such as MTT or alamarBlue) to establish relationships between CCL4 expression and cell survival.

This approach has revealed that CCL4 expression is induced prior to the manifestation of cytotoxicity in neural cells exposed to MeHg, suggesting a potential adaptive or protective response .

What are the primary signaling pathways activated by Recombinant Mouse CCL4?

Recombinant Mouse CCL4 primarily activates signaling through its receptor CCR5, initiating several downstream pathways:

• G-protein coupled signaling: Upon binding to CCR5, CCL4 activates G-protein-mediated signaling cascades that lead to:

  • Calcium mobilization from intracellular stores

  • Activation of phospholipase C (PLC)

  • Generation of inositol triphosphate (IP3) and diacylglycerol (DAG)

• Rac1/Cdc42 pathway: CCL4 mediates lymphocyte adhesion through activation of Rac1/Cdc42 signaling, which regulates cytoskeletal reorganization essential for cell migration and adhesion.

• JAK/STAT signaling: CCL4 can induce activation of JAK/STAT pathways, particularly STAT1 and STAT3, contributing to inflammatory gene expression.

• MAPK pathways: Activation of mitogen-activated protein kinase (MAPK) cascades, including ERK1/2, p38, and JNK, mediates various cellular responses to CCL4 including cell survival, differentiation, and cytokine production.

These signaling mechanisms collectively contribute to CCL4's roles in immune cell recruitment, inflammatory responses, and potential neuroprotective functions .

What is the role of CCL4 in neuroprotection against methylmercury toxicity?

Research demonstrates that CCL4 plays a significant protective role against methylmercury (MeHg) toxicity in the central nervous system through several mechanisms:

• Induced expression prior to damage: Studies show that CCL4 expression is selectively upregulated in the brain of MeHg-administered mice prior to the manifestation of neuronal damage, suggesting an adaptive response.

• Direct neuroprotective effects: Recombinant CCL4 attenuates MeHg-induced cytotoxicity in primary mouse neuron cultures in a dose-dependent manner, with significant protection observed at concentrations as low as 10 ng/mL.

• Endogenous protective mechanism: CCL4 expression is induced in C17.2 neural stem cells prior to MeHg-induced cytotoxicity, indicating that endogenous CCL4 production may serve as a protective mechanism against MeHg toxicity.

• Essential for neuronal survival: Knockdown of CCL4 expression enhances MeHg cytotoxicity in C17.2 cells, confirming that endogenous CCL4 expression is critical for neuronal survival during MeHg exposure.

These findings collectively suggest that CCL4 induction represents an endogenous neuroprotective mechanism against MeHg toxicity, potentially through activation of pro-survival signaling pathways and/or modulation of inflammatory responses .

How can researchers establish CCL4 knockdown models for functional studies?

To establish effective CCL4 knockdown models for functional studies, researchers can follow this methodological approach:

• Selection of knockdown strategy:

  • siRNA-mediated knockdown: Transient suppression suitable for short-term experiments

  • shRNA-mediated knockdown: More stable suppression for longer-term studies

  • CRISPR/Cas9-mediated gene editing: For complete gene knockout studies

• Design of targeting sequences:

  • Design multiple siRNA/shRNA sequences targeting different regions of the CCL4 mRNA

  • For CRISPR/Cas9, design guide RNAs targeting early exons to ensure functional knockout

• Validation of knockdown efficiency:

  • Quantitative RT-PCR to measure reduction in CCL4 mRNA levels

  • ELISA or Western blotting to confirm reduction in CCL4 protein expression

  • Functional assays (e.g., chemotaxis assays) to verify reduced CCL4 activity

• Experimental design considerations:

  • Include appropriate controls (scrambled siRNA, non-targeting shRNA, or Cas9 without guide RNA)

  • Establish time course of knockdown to determine optimal experimental window

  • Consider potential compensatory mechanisms (upregulation of related chemokines)

• Application to functional studies:

  • Combine knockdown with cellular stress models (e.g., MeHg exposure) to assess CCL4's protective role

  • Monitor multiple endpoints including cell viability, morphology, and downstream signaling

Research has demonstrated that knockdown of CCL4 in C17.2 neural stem cells enhances sensitivity to MeHg cytotoxicity, confirming the protective role of endogenous CCL4 expression .

What factors should be considered when comparing results from different recombinant CCL4 preparations?

When comparing results obtained using different recombinant CCL4 preparations, researchers should consider several critical factors:

• Expression system variations:

  • E. coli-derived proteins may differ from mammalian cell-derived proteins in post-translational modifications

  • Different expression systems may yield proteins with varying levels of biological activity

• Tag influence:

  • The presence of tags (e.g., His-tag) can affect protein folding, receptor binding, and biological activity

  • Tagged proteins (11.8 kDa for His-tagged CCL4) versus tag-free proteins (7.8 kDa) may show different behaviors in experimental systems

• Purity considerations:

  • Variations in protein purity (e.g., >90% versus >97%) can impact experimental outcomes

  • Contaminants may introduce confounding biological activities

• Endotoxin levels:

  • Different preparations may contain varying levels of endotoxin

  • High endotoxin levels can independently activate immune cells and confound CCL4-specific effects

• Reconstitution and storage conditions:

  • Different buffer compositions can affect protein stability and activity

  • Variations in glycerol concentration (5-50%) may impact protein behavior

  • Freeze-thaw cycles can progressively reduce biological activity

• Lot-to-lot variations:

  • Even within the same supplier, different production lots may exhibit subtle differences in activity

  • Batch testing for consistent bioactivity is recommended for critical experiments

To maximize reproducibility, researchers should maintain consistent sourcing of recombinant proteins when possible, or perform careful cross-validation when switching between preparations .

How can researchers differentiate between direct and indirect effects of CCL4 in complex experimental systems?

Differentiating between direct and indirect effects of CCL4 in complex experimental systems requires strategic experimental design:

• Receptor blocking studies:

  • Use CCR5-specific antagonists to block direct CCL4 signaling

  • Compare outcomes of CCL4 treatment with and without receptor blockade

  • Effects that persist despite receptor blockade likely represent indirect mechanisms

• Cell-specific approaches:

  • Use isolated cell populations to identify cell-autonomous responses

  • Compare CCL4 effects in monocultures versus co-culture systems

  • Employ conditioned media experiments to identify secreted mediators

• Temporal analysis:

  • Establish detailed time courses of responses following CCL4 treatment

  • Early responses (minutes to hours) more likely represent direct effects

  • Delayed responses (hours to days) may involve secondary mediators

• Signaling pathway dissection:

  • Use specific inhibitors of downstream signaling pathways

  • Identify which cellular outcomes are linked to specific signaling modules

  • Map the hierarchy of signaling events following CCL4 exposure

• Genetic approaches:

  • Compare CCL4 effects in wild-type versus CCR5-knockout systems

  • Use inducible expression systems to control timing of CCL4 production

  • Employ targeted gene deletions in specific cell populations to determine cellular origins of responses

• Combined in vitro and in vivo approaches:

  • Validate in vitro findings in more complex in vivo systems

  • Use tissue-specific knockout or knockdown models to isolate cell-specific contributions

By systematically applying these approaches, researchers can build a comprehensive understanding of both direct receptor-mediated effects and indirect effects mediated by secondary signals or cell-cell interactions .

What quantitative data supports the neuroprotective role of CCL4?

Experimental evidence demonstrating CCL4's neuroprotective effects against methylmercury toxicity includes the following quantitative data:

Table 1: Dose-dependent neuroprotection by recombinant CCL4 in primary cerebellar granule cells exposed to methylmercury

These findings demonstrate that recombinant CCL4 significantly attenuates MeHg-induced cytotoxicity in primary mouse neuron cultures in a dose-dependent manner, with protection observed at concentrations as low as 10 ng/mL .

What are the key experimental controls required when studying CCL4 in neuronal systems?

When investigating CCL4's functions in neuronal systems, the following key experimental controls should be implemented:

• Vehicle controls:

  • Solvent-only controls for CCL4 (typically buffer containing the same components without protein)

  • Vehicle controls for any neurotoxic agents (e.g., MeHg dissolved in the appropriate vehicle)

• Concentration controls:

  • Dose-response experiments with varying CCL4 concentrations (typically 10-100 ng/mL)

  • Include both suboptimal and potentially supraphysiological concentrations

• Specificity controls:

  • Include related chemokines (e.g., CCL2) to assess specificity of observed effects

  • Use heat-inactivated CCL4 to confirm that effects require functional protein

• Temporal controls:

  • Vary pre-treatment times with CCL4 before neurotoxic challenge

  • Establish time course of neurotoxic effects with and without CCL4

• Cell type controls:

  • Compare effects in neurons, glial cells, and mixed cultures

  • Use cell-type specific markers (e.g., NeuN for neurons) to identify responding populations

• Mechanistic controls:

  • CCR5 receptor antagonists to block CCL4-specific signaling

  • Pathway inhibitors to identify key downstream mediators

These controls help establish the specificity, dose-dependency, and mechanism of CCL4-mediated effects in neuronal systems, minimizing the risk of experimental artifacts or misinterpretation of results .