Recombinant Human Eotaxin protein (CCL11) (Active)

What is the amino acid sequence and molecular weight of recombinant human Eotaxin?

Recombinant human Eotaxin protein encompasses amino acids 24-97 of the full sequence with the following sequence: GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP. The predicted molecular weight is approximately 8421.95 Da as determined by ESI-TOF analysis, with the observed weight being 8421.91 Da. Additional masses at 8624.00 and 8989.00 Da may be detected due to residual O-glycans present on the protein .

What structural family does Eotaxin belong to, and what are its known post-translational modifications?

Eotaxin (CCL11) belongs to the intercrine beta (chemokine CC) family. Its principal post-translational modification is O-linked glycosylation, consisting of a Gal-GalNAc disaccharide which can be further modified with up to two sialic acid residues . These modifications can impact the protein's biological activity and should be considered when designing experiments, particularly those examining receptor interactions or signaling pathways.

What are the alternative nomenclatures for Eotaxin that appear in scientific literature?

When reviewing literature, researchers should be aware of multiple designations: SCYA11, CCL11, Eotaxin, C-C motif chemokine 11, Eosinophil chemotactic protein, and Small-inducible cytokine A11 . Using consistent terminology in publications and understanding these alternative names is critical for comprehensive literature searches and proper citation of previous research.

How do expression systems affect the properties of recombinant Eotaxin protein?

Recombinant human Eotaxin is typically produced in either prokaryotic (E. coli) or eukaryotic (HEK 293) expression systems. E. coli-expressed protein may lack post-translational modifications but offers high yield with >97% purity and endotoxin levels <1 EU/μg . In contrast, HEK 293-expressed Eotaxin better represents naturally occurring protein with appropriate glycosylation patterns, offering >95% purity with endotoxin levels ≤0.005 EU/μg . The choice of expression system should align with experimental requirements, particularly when biological activity dependent on glycosylation is being studied.

What purification methods are most effective for obtaining high-purity Eotaxin protein?

High-purity Eotaxin typically undergoes multi-step purification protocols involving affinity chromatography, followed by ion exchange chromatography and size exclusion chromatography. These methods can achieve >95-97% purity as verified by SDS-PAGE analysis . For applications requiring extremely low endotoxin content, additional endotoxin removal steps such as Triton X-114 phase separation or polymyxin B affinity may be incorporated into the purification workflow to achieve levels as low as 0.005 EU/μg .

How can researchers verify the authenticity and activity of recombinant Eotaxin preparations?

Authentication requires a multi-analytical approach:

• SDS-PAGE for molecular weight and purity verification

• HPLC analysis for homogeneity assessment

• Mass spectrometry (ESI-TOF) for precise molecular weight determination

• Functional assays measuring chemotactic activity toward eosinophils

• Receptor binding assays confirming interaction with CCR3

Active protein typically demonstrates dose-dependent eosinophil migration in Boyden chamber assays and specific binding to CCR3-expressing cells .

What critical quality control parameters should be assessed when working with Eotaxin protein?

Critical quality control parameters include:

Verification of these parameters ensures experimental reproducibility and biological relevance .

How do different analytical techniques compare in assessing Eotaxin quality and functionality?

Each analytical technique provides complementary information:

• SDS-PAGE: Rapid assessment of purity and approximate molecular weight but limited resolution

• HPLC: Superior resolution for detecting closely related contaminants or degradation products

• Mass Spectrometry: Precise molecular weight determination and identification of post-translational modifications

• Functional Assays: Direct measurement of biological activity but higher variability

• ELISA: Quantitative determination with high sensitivity but dependent on antibody quality

A comprehensive quality assessment should incorporate multiple techniques rather than relying on a single method .

What is the recommended approach for quantifying Eotaxin in biological samples?

For accurate quantification in biological samples such as serum, plasma, or cell culture supernatants, a validated ELISA methodology is recommended. Commercially available kits show dose-response curves that parallel standard curves generated with recombinant protein, confirming their suitability for determining relative mass values of natural human Eotaxin . For complex biological matrices, sample pre-treatment and validation of dilution linearity are essential to minimize matrix effects and ensure accurate measurements.

How does Eotaxin function in allergic inflammatory pathways?

Eotaxin directly promotes eosinophil accumulation in response to allergens, representing a key feature of allergic inflammatory reactions . The protein binds to CCR3 receptors expressed predominantly on eosinophils, basophils, and Th2 cells, initiating chemotactic responses through G-protein-coupled signaling pathways . This chemokine-receptor interaction activates intracellular calcium mobilization, actin polymerization, and directional cell migration, ultimately contributing to tissue eosinophilia characteristic of allergic conditions such as asthma, atopic dermatitis, and allergic rhinitis.

What experimental models effectively demonstrate Eotaxin's role in eosinophil recruitment?

Several experimental approaches have demonstrated Eotaxin's chemotactic activity:

• In vitro Transwell migration assays: Using purified eosinophils or CCR3-expressing cell lines to quantify directed cell movement in response to concentration gradients of recombinant Eotaxin

• Ex vivo tissue explant models: Measuring eosinophil infiltration into tissue samples following Eotaxin exposure

• In vivo models: Administration of recombinant Eotaxin in animal models induces rapid and selective eosinophil recruitment to injection sites

• Allergen challenge models: Monitoring Eotaxin expression and eosinophil recruitment following allergen exposure in sensitized animals

These models provide complementary insights into Eotaxin's chemotactic function across different levels of biological complexity .

How can Eotaxin research contribute to understanding non-allergic conditions?

Beyond allergic inflammation, Eotaxin has emerging roles in:

• Neurodegenerative disorders: Elevated CCL11 levels in cerebrospinal fluid have been associated with chronic traumatic encephalopathy, suggesting potential roles in neuroinflammation

• Aging processes: Increased plasma CCL11 concentrations correlate with cognitive decline in older adults, with geographical variations between rural and urban dwellers suggesting environmental influences

• Pulmonary fibrosis: Bronchoalveolar lavage fluid from patients with mustard gas-induced pulmonary fibrosis shows altered CCL11 levels, indicating involvement in fibrosing lung diseases

• COVID-19 pathophysiology: Studies of asymptomatic and symptomatic SARS-CoV-2-infected individuals have revealed distinct immune signatures involving CCL11, suggesting roles in COVID-19 disease progression

These diverse applications highlight the expanding significance of Eotaxin research beyond classical allergic conditions.

What are the known interactions between Eotaxin and other inflammatory mediators in complex biological systems?

Eotaxin functions within a complex network of inflammatory mediators. Key interactions include:

• Synergistic effects with IL-5: While Eotaxin primarily mediates eosinophil recruitment, IL-5 enhances eosinophil development and survival, creating a cooperative mechanism that amplifies tissue eosinophilia

• Cross-regulation with IL-13: Research has shown that IL-13 induces Eotaxin expression in airway epithelial cells and fibroblasts, while IL-13 receptor alpha2 levels modulate this response

• Relationship with TNF-α: TNF-α can upregulate Eotaxin production in various cell types, contributing to remodeling processes in airway smooth muscle cells

• microRNA regulation: Studies have identified specific microRNAs (including miR-124-3p and miR-31) that modulate Eotaxin expression and associated inflammatory pathways

Understanding these complex interactions is essential for developing targeted therapeutic strategies that address multiple components of eosinophilic inflammation.

How do post-translational modifications impact Eotaxin's biological activity?

The O-linked glycan consisting of a Gal-GalNAc disaccharide modified with up to two sialic acid residues significantly influences Eotaxin's:

• Receptor binding affinity: Glycosylation patterns can alter the three-dimensional structure of the protein, potentially affecting its interaction with CCR3 receptors

• Circulatory half-life: Sialic acid residues can protect the protein from degradation and clearance, extending its biological activity in vivo

• Tissue diffusion properties: The presence and composition of glycans influence the protein's ability to establish chemotactic gradients within tissue matrices

• Immunogenicity: Different glycoforms may exhibit varying immunogenic properties, particularly in the context of recombinant protein administration

These considerations are particularly important when comparing E. coli-expressed (non-glycosylated) versus HEK 293-expressed (glycosylated) recombinant proteins for specific applications.

What advanced techniques can provide insights into the structural biology of Eotaxin-receptor interactions?

Advanced structural biology techniques to investigate Eotaxin-CCR3 interactions include:

• X-ray crystallography: Determining the atomic structure of Eotaxin alone or in complex with CCR3 peptides

• Nuclear Magnetic Resonance (NMR) spectroscopy: Characterizing protein dynamics and identifying specific residues involved in receptor binding

• Cryo-electron microscopy: Visualizing the full-length CCR3 receptor in complex with Eotaxin

• Molecular dynamics simulations: Modeling the conformational changes during chemokine-receptor binding and activation

• Surface plasmon resonance (SPR): Quantifying binding kinetics and affinity constants between Eotaxin and CCR3

• Hydrogen-deuterium exchange mass spectrometry: Mapping protein interfaces involved in the binding interaction

These approaches can identify critical binding epitopes and inform structure-based drug design targeting the Eotaxin-CCR3 axis.

What are the key considerations for designing dose-response experiments with recombinant Eotaxin?

Effective dose-response experiments should account for:

• Concentration range selection: Typically, a logarithmic concentration series spanning 0.1-100 ng/mL for in vitro studies, with narrowing of ranges based on initial results

• Appropriate controls: Including both negative controls (buffer only) and positive controls (established chemoattractants like IL-8 or fMLP)

• Receptor saturation effects: At high concentrations, chemokines can induce receptor desensitization, creating bell-shaped rather than sigmoidal dose-response curves

• Incubation time optimization: Different biological responses (calcium flux, chemotaxis, gene expression) require distinct time points for measurement

• Vehicle and carrier protein considerations: Ensure vehicle composition and carrier protein concentration remain constant across all dosage points

Properly designed dose-response studies enable accurate EC₅₀ determination and facilitate comparison between different Eotaxin preparations or experimental conditions .

How should researchers address variability in Eotaxin detection across different biological samples?

Strategies to manage variability in Eotaxin quantification include:

• Sample-specific optimization: Different matrices (serum, plasma, BAL fluid, cell culture supernatants) require specific dilution protocols and pre-treatment steps

• Standard curve matrix matching: Preparing standard curves in matrices similar to test samples improves quantification accuracy

• Spike recovery validation: Adding known quantities of recombinant Eotaxin to samples validates detection efficiency in specific matrices

• Inter-laboratory standardization: Utilizing common reference materials and standardized protocols reduces systematic variations between research groups

• Consideration of binding proteins: Accounting for potential binding proteins in biological samples that may interfere with detection

These approaches are particularly important when comparing Eotaxin levels across diverse sample types, as demonstrated in studies measuring CCL11 in serum, plasma, urine, and bronchoalveolar lavage fluid .

What strategies can improve reproducibility in functional assays using recombinant Eotaxin?

To enhance reproducibility in functional assays:

• Standardized cell sources: Use well-characterized cell lines or primary cells with defined isolation and culture protocols

• Controlled storage conditions: Implement consistent protein aliquoting, storage temperatures, and avoid freeze-thaw cycles

• Validated activity measurements: Confirm biological activity of each new lot using established assays before complex experiments

• Normalized reporting: Express results relative to positive controls to account for day-to-day variability

• Consistent experimental timing: Standardize the duration of cell starvation, stimulation, and measurement periods

• Environmental parameter control: Maintain consistent temperature, humidity, and CO₂ levels during assay performance

• Detailed protocol documentation: Record all experimental variables including reagent sources, lot numbers, and exact timings

Implementation of these practices significantly reduces variability in chemotaxis, calcium flux, and cell signaling assays involving Eotaxin .

How can researchers address common challenges in recombinant Eotaxin protein handling and storage?

Common challenges and their solutions include:

• Activity loss during storage:

  • Store as single-use aliquots at -80°C

  • Add carrier proteins (0.1-1% BSA) to prevent adsorption to container surfaces

  • Avoid repeated freeze-thaw cycles

• Protein aggregation:

  • Centrifuge briefly before use to remove potential aggregates

  • Filter through 0.22 μm filters for critical applications

  • Maintain recommended pH and ionic strength conditions

• Variable biological responses:

  • Characterize each new lot before use in complex experiments

  • Include internal standards for normalization between experiments

  • Consider the impact of expression systems on protein activity

• Endotoxin contamination:

  • Select preparations with certified low endotoxin levels (<0.005 EU/μg)

  • Include polymyxin B controls in sensitive cell assays

  • Consider endotoxin removal for sensitive applications

These strategies help maintain protein integrity and functional consistency across experiments .

What approaches can resolve discrepancies between different Eotaxin detection methods?

When facing discrepancies between detection methods:

• Method-specific biases:

  • ELISA may detect both free and receptor-bound Eotaxin

  • Mass spectrometry distinguishes different isoforms and modifications

  • Bioassays measure only functionally active protein

• Resolution strategies:

  • Cross-validate samples using orthogonal detection methods

  • Characterize what each assay actually measures (total vs. active protein)

  • Standardize sample collection and processing protocols

  • Consider epitope accessibility in antibody-based methods

• Reporting practices:

  • Explicitly state detection method limitations in publications

  • Report raw values alongside normalized data when possible

  • Include assay validation parameters (sensitivity, specificity, range)

These approaches help reconcile apparently conflicting results from different measurement techniques .

How should researchers interpret differences between recombinant and native Eotaxin in experimental systems?

When interpreting differences between recombinant and native Eotaxin:

• Source-dependent variations:

  • E. coli-expressed protein lacks glycosylation present in native Eotaxin

  • HEK 293-expressed protein better resembles native glycosylation patterns

  • Natural Eotaxin may exist as multiple isoforms in biological samples

• Functional considerations:

  • Compare dose-response curves between recombinant and native preparations

  • Validate that parallel response curves indicate similar biological activity

  • Consider that differential glycosylation may affect receptor binding kinetics

• Experimental design implications:

  • Include both recombinant and native sources when possible

  • Use recombinant protein for standardization and quantification

  • Consider native sources for physiological relevance

Results obtained measuring natural human Eotaxin typically show dose-response curves parallel to standard curves obtained using recombinant proteins, indicating that recombinant preparations are suitable for determining relative mass values for natural human Eotaxin .