Recombinant Danio rerio Collectin-12 (colec12)
Description
Introduction to Collectin-12 (colec12)
Collectin-12 (colec12) in Danio rerio (zebrafish) is a member of the collectin subfamily that plays essential roles in innate immunity and developmental processes. Collectins are characterized by their collagen-like domains and C-type lectin domains, which enable them to recognize and bind to specific molecular patterns on pathogens and modified self-structures . The zebrafish colec12 gene is located on chromosome 2 and encodes a protein that functions in pattern recognition and low-density lipoprotein particle binding .
Zebrafish have become an increasingly important model organism for studying vertebrate development, disease, and toxicology due to their genetic tractability, transparent embryos, and rapid development . This makes them particularly valuable for investigating proteins like colec12, whose functions span multiple biological systems.
The orthologous relationship between zebrafish colec12 and human COLEC12 suggests conserved functions across vertebrates, making findings in the zebrafish model potentially translatable to human biology and disease mechanisms . This evolutionary conservation underscores the importance of studying colec12 in zebrafish as a means to understand broader principles of collectin biology across species.
Functional Properties of colec12
Danio rerio colec12 demonstrates multiple functional properties that contribute to its significant biological roles. According to available research, the protein is involved in pattern recognition, low-density lipoprotein particle binding, and vasculogenesis . These functions position colec12 at the intersection of immunity and development in zebrafish.
As a pattern recognition receptor, colec12 participates in the innate immune system's ability to recognize and respond to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) . This immune surveillance function represents a critical first-line defense mechanism against infections.
The functional properties of colec12 include:
Expression studies have demonstrated that colec12 is present in several structures in zebrafish, including the cardiovascular system, epidermis, mesoderm, musculature system, and periderm . This broad expression pattern indicates versatile roles in different tissues and developmental stages, suggesting that colec12 may have tissue-specific functions throughout zebrafish development.
The involvement of colec12 in vasculogenesis is particularly significant, as it points to a role in the formation of blood vessels during embryonic development . This function may be linked to the protein's ability to interact with extracellular matrix components and its potential participation in cell-cell and cell-matrix signaling pathways critical for proper vascular development.
Recombinant Production of Danio rerio colec12
Recombinant Danio rerio colec12 protein production provides researchers with a valuable tool for studying this important molecule. The recombinant protein is typically produced as a full-length protein (1-720 amino acids) with an N-terminal His tag, expressed in E. coli expression systems .
The specifications and characteristics of commercially available recombinant Danio rerio colec12 are detailed in the following table:
| Parameter | Specification |
|---|---|
| Catalog Number | RFL18326DF |
| Source | E. coli |
| Tag | His (N-terminal) |
| Protein Length | Full Length (1-720 amino acids) |
| Form | Lyophilized powder |
| Purity | Greater than 90% as determined by SDS-PAGE |
| Applications | SDS-PAGE |
| Storage | Store at -20°C/-80°C, avoid repeated freeze-thaw cycles |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Reconstitution | In deionized sterile water to 0.1-1.0 mg/mL, with recommended 5-50% glycerol for long-term storage |
The production process involves expressing the protein in bacterial systems, followed by purification steps that typically utilize the affinity of the His-tag for metal ions. The resulting high-purity protein (>90%) can be used for various research applications . The lyophilized format provides stability for shipping and storage, while the reconstitution guidelines ensure proper preparation for experimental use.
The N-terminal His-tag facilitates protein purification and detection without significantly altering the functional properties of the protein. The full-length structure ensures that all functional domains are present, allowing for comprehensive studies of protein-protein interactions, binding activities, and structural analyses .
Applications and Research Significance
Recombinant Danio rerio colec12 has significant applications across multiple research domains, including developmental biology, immunology, and comparative genomics. The availability of purified recombinant protein enables detailed investigations of its structure, function, and molecular interactions.
In developmental biology, colec12's role in vasculogenesis makes it a valuable target for studies of blood vessel formation and cardiovascular development in zebrafish . Researchers can use the recombinant protein to study binding partners involved in vessel formation, to develop antibodies for tracking protein expression during development, and to perform functional assays assessing its role in various developmental processes.
For immunological research, colec12's function as a pattern recognition receptor highlights its relevance to studies of innate immunity in zebrafish . The recombinant protein can be employed to investigate binding specificity to various pathogen-associated molecular patterns, to examine interactions with other components of the immune system, and to develop models of immune response and regulation.
The zebrafish model offers unique advantages for these studies, including genetic tractability, transparent embryos for real-time imaging, and rapid development . These features allow researchers to observe the effects of colec12 manipulation in vivo and to track developmental and immunological processes with high temporal and spatial resolution.
In comparative genomics, the orthologous relationship between zebrafish colec12 and human COLEC12 provides an opportunity to investigate evolutionarily conserved functions . The recombinant protein facilitates comparative studies that can identify structural and functional similarities and differences between zebrafish and human collectins, potentially revealing insights relevant to human health and disease.
Comparative Analysis with Human COLEC12
Human COLEC12 and Danio rerio colec12 share significant homology, reflecting their orthologous relationship and suggesting conserved functions across vertebrate evolution . This comparative perspective provides valuable insights into both conserved and species-specific features of collectin biology.
Both human COLEC12 and zebrafish colec12 belong to the collectin subfamily and share similar domain structures, including C-type lectin domains and collagen-like regions . They are both predicted to function as pattern recognition receptors and to bind low-density lipoprotein particles, suggesting conservation of core functions .
Human COLEC12 has been documented to function as a scavenger receptor involved in the recognition and clearance of various ligands, including modified lipoproteins and pathogen-associated molecular patterns . Additionally, human COLEC12 has been shown to bind fibrillar β-amyloid protein , suggesting potential involvement in neurodegenerative disease processes. Whether zebrafish colec12 shares these specific functions remains to be fully elucidated, but the structural similarity suggests potential functional conservation.
The table below summarizes key similarities and differences between human COLEC12 and zebrafish colec12:
This comparative analysis highlights the potential of zebrafish as a model for studying conserved functions of collectins across vertebrates and for investigating the role of these proteins in development, immunity, and potentially in disease processes relevant to human health.
Current Research Trends
Current research on Danio rerio colec12 is situated within broader trends in zebrafish research, particularly as it relates to developmental biology, immunology, and toxicology. The zebrafish model has gained significant attention for its utility in these fields, providing valuable insights that may be applicable to human health and disease.
In developmental biology, investigations of colec12 contribute to understanding the molecular mechanisms of vasculogenesis and cardiovascular system formation . Researchers are exploring how this protein interacts with other molecules to guide the formation of blood vessels during embryonic development, a process with implications for understanding both normal development and vascular disorders.
In immunological research, the study of colec12 as a pattern recognition molecule aligns with broader investigations of innate immunity in zebrafish . These studies aim to elucidate the evolution and conservation of immune mechanisms across vertebrates and to identify potential therapeutic targets for immune-related disorders. The collectin family's role in recognizing pathogen-associated molecular patterns makes them particularly relevant to research on infectious diseases and immune responses.
In toxicology, zebrafish have emerged as an important in vivo model for assessing the effects of various compounds on development and physiological processes . Zebrafish toxicology studies offer advantages including rapid development, transparent embryos for real-time observation, and genetic tractability that makes them suitable for high-throughput screening applications.
The availability of recombinant proteins like colec12, combined with advanced genetic tools such as CRISPR-Cas9 for gene editing, facilitates comprehensive functional studies. These approaches allow researchers to investigate the roles of specific domains within the protein, to examine the effects of mutations or modifications, and to identify interaction partners and signaling pathways that may be relevant to human health and disease.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you require a specific format, please indicate your preference when placing your order. We will fulfill your request accordingly.
Note: All our proteins are shipped with standard blue ice packs. If dry ice shipment is required, please communicate with us beforehand as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What expression systems are suitable for producing recombinant Danio rerio Collectin-12?
Two primary expression systems have been documented for recombinant Danio rerio Collectin-12 production:
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E. coli expression system: Recombinant full-length Danio rerio Collectin-12 (1-720aa) has been successfully expressed in E. coli with an N-terminal His-tag . This system offers advantages for high protein yield and simplified purification protocols.
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Mammalian expression system: For studies requiring post-translational modifications similar to native protein, mammalian expression systems might be preferable. The search results show that mammalian systems such as Flp-In™-CHO cells have been used successfully for other collectins like human CL-12 . This approach allows for proper oligomerization and glycosylation.
The choice between these systems depends on research requirements. E. coli is suitable for structural studies and applications not dependent on glycosylation, while mammalian systems are preferable when native-like post-translational modifications are essential for functional studies.
How can researchers verify the identity and integrity of recombinant Danio rerio Collectin-12?
Verification of recombinant Danio rerio Collectin-12 typically involves multiple analytical approaches:
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SDS-PAGE analysis: Under both reducing and non-reducing conditions to assess molecular weight and oligomerization status. Properly produced recombinant zebrafish Collectin-12 should show >90% purity by SDS-PAGE .
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Western blot analysis: Using either anti-His antibodies (for tagged versions) or specific anti-Collectin-12 antibodies. When analyzing recombinant collectins under non-reducing conditions, one should expect to observe a ladder-like pattern representing monomers, dimers, and trimers, while reducing conditions should show predominantly monomeric forms .
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Mass spectrometry: For accurate molecular weight determination and verification of amino acid sequence.
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N-terminal sequencing: To confirm the correct processing of the protein.
A comprehensive verification protocol should include multiple methods to ensure both identity and structural integrity before proceeding with functional studies.
What is the recommended protocol for producing His-tagged recombinant Danio rerio Collectin-12 in E. coli?
Based on the available information, a general protocol for producing His-tagged recombinant Danio rerio Collectin-12 in E. coli would include:
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Vector construction: Design an expression construct containing the full-length (1-720aa) or desired fragment of zebrafish colec12 with an N-terminal His-tag .
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Transformation: Transform the expression construct into an appropriate E. coli strain optimized for recombinant protein expression.
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Culture and induction: Grow transformed bacteria to mid-log phase, then induce protein expression with an appropriate inducer (typically IPTG for T7-based expression systems).
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Cell harvest and lysis: Collect bacterial cells by centrifugation and lyse using methods that preserve protein integrity (sonication, French press, or chemical lysis).
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Purification: Purify using immobilized metal affinity chromatography (IMAC), taking advantage of the His-tag.
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Quality control: Verify purity (>90%) using SDS-PAGE and other analytical methods .
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Lyophilization: The final product is often prepared as a lyophilized powder for long-term stability .
For applications requiring higher purity or specific structural properties, additional purification steps such as size exclusion chromatography or ion exchange chromatography may be necessary.
What purification strategies maximize yield and activity of recombinant Danio rerio Collectin-12?
Effective purification of recombinant Danio rerio Collectin-12 requires strategies that balance yield with preservation of protein activity:
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Affinity chromatography: For His-tagged constructs, nickel or cobalt-based IMAC is the primary purification method, allowing selective isolation of the target protein .
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Buffer optimization: Collectins have specific structural requirements; therefore, buffer composition is critical. Tris/PBS-based buffers at pH 8.0 have been documented as suitable for storage of purified zebrafish Collectin-12 .
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Stabilizing additives: The addition of stabilizers such as trehalose (6%) helps maintain protein structure during purification and storage processes .
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Gentle elution conditions: Using imidazole gradients rather than step elution can help preserve protein structure during IMAC purification.
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Prevention of aggregation: Careful control of protein concentration and temperature during purification helps prevent aggregation.
When scaling up production, these parameters must be carefully monitored and adjusted to maintain consistency in yield and activity.
What storage conditions are optimal for maintaining the activity of purified recombinant Danio rerio Collectin-12?
Optimal storage conditions for recombinant Danio rerio Collectin-12 include:
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Temperature: Store at -20°C/-80°C for long-term storage, with -80°C preferred for maximum stability .
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Aliquoting: Division into single-use aliquots is crucial to avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity .
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Cryoprotectants: Addition of glycerol (recommended final concentration of 50%) or other cryoprotectants helps prevent freeze-thaw damage .
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Reconstitution guidelines: When using lyophilized protein, reconstitution should be in deionized sterile water to a concentration of 0.1-1.0 mg/mL .
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Working stock handling: For short-term use, working aliquots can be maintained at 4°C for up to one week .
Adherence to these storage recommendations is essential for maintaining protein activity and structural integrity, particularly for functional studies.
How can recombinant Danio rerio Collectin-12 be used to study neural crest cell development?
Recombinant Danio rerio Collectin-12 can serve as a valuable tool in neural crest cell (NCC) development studies:
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Expression pattern analysis: By comparing the spatiotemporal expression of colec12 with established NCC markers like sox10, researchers can determine whether colec12 is expressed in specific NCC lineages. The comprehensive single-cell transcriptomic atlas of sox10-expressing and sox10-derived populations in zebrafish provides a reference framework for such analyses .
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Functional studies: Recombinant colec12 can be used in gain-of-function experiments to assess its effects on NCC migration, differentiation, or survival. The atlas identifying eight major classes of cells derived from sox10-expressing progenitors (mesenchyme, NCC, neural, neuronal, glial, pigment, muscle, and otic) provides potential cellular contexts for such studies .
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Protein interaction studies: Using tagged recombinant colec12, researchers can identify binding partners in neural crest cells through pull-down assays, potentially revealing novel signaling pathways relevant to NCC development.
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Comparative analyses: Studies comparing the effects of wild-type versus mutant forms of recombinant colec12 can help elucidate the protein's functional domains important for NCC development.
The interactive cell browser resource (https://zebrafish-neural-crest-atlas.cells.ucsc.edu/) containing the comprehensive cell type atlas of posterior sox10-expressing cells can help researchers identify specific developmental stages and cell populations for these investigations .
What experimental approaches are recommended for studying the function of Collectin-12 in zebrafish development?
Several experimental approaches are recommended for investigating Collectin-12 function in zebrafish development:
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Morpholino knockdown and CRISPR/Cas9 gene editing: These complementary approaches allow for loss-of-function studies to determine the developmental consequences of colec12 deficiency.
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Transgenic rescue experiments: Following knockdown or knockout, phenotypes can be rescued using wild-type or mutant forms of recombinant colec12 to map functional domains.
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Lineage tracing: Using the Tg(-4.9sox10:EGFP) transgenic line (referred to as sox10:GFP) in conjunction with colec12 manipulation can help track the fate of neural crest derivatives .
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Hybridization Chain Reaction (HCR): This technique has been successfully used to validate spatiotemporal expression patterns of various subtypes in the neural crest lineage atlas and can be applied to study colec12 expression in relation to other developmental markers .
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Single-cell transcriptomics: Following the methodologies described in the neural crest atlas studies, researchers can assess the impact of colec12 manipulation on cell fate decisions at single-cell resolution .
These approaches can be integrated to provide a comprehensive understanding of Collectin-12's role in zebrafish development, particularly in neural crest-derived tissues.
How does oligomerization affect the functional properties of recombinant Danio rerio Collectin-12?
Oligomerization is a critical determinant of collectin function, including Collectin-12:
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Structural implications: Similar to human CL-12, which forms monomers, dimers, and trimers, zebrafish Collectin-12 likely forms oligomeric structures that influence its biological activity . This oligomerization is typically maintained during secretion, suggesting it is essential for proper function .
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Binding properties: Oligomerization of collectins dictates their binding properties, with higher-order oligomers typically demonstrating enhanced avidity for their ligands . For recombinant Danio rerio Collectin-12, the preservation of these oligomeric states is likely crucial for maintaining native-like binding characteristics.
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Complement activation: The oligomeric state of collectins influences their ability to activate the complement system . Studies with recombinant human CL-12 have shown that its oligomerization pattern influences this function, and similar principles may apply to zebrafish Collectin-12.
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Experimental considerations: When designing experiments with recombinant Danio rerio Collectin-12, researchers should assess oligomerization status through methods such as native PAGE or size exclusion chromatography. Comparison of reducing versus non-reducing SDS-PAGE can provide initial insights into the presence of disulfide-stabilized oligomers .
Understanding and preserving the natural oligomerization properties of recombinant Danio rerio Collectin-12 is essential for experiments intended to recapitulate physiological functions.
What are common challenges in producing soluble recombinant Danio rerio Collectin-12?
Production of soluble recombinant Danio rerio Collectin-12 presents several challenges:
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Proper folding: The complex domain structure of Collectin-12, including its collagen-like domain with Gly-X-Y repeats, can lead to folding difficulties, especially in prokaryotic expression systems.
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Maintaining oligomeric structure: Ensuring the recombinant protein forms the correct oligomeric assemblies (monomers, dimers, and trimers) observed in native conditions .
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Solubility issues: The collagen-like domain can promote aggregation, particularly at higher concentrations.
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Post-translational modifications: If produced in E. coli, the protein will lack glycosylation that might be present in the native protein, potentially affecting folding and function.
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Proteolytic degradation: The extended structure of collectins can make them susceptible to proteolysis during expression and purification.
To address these challenges, researchers can:
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Consider mammalian expression systems for complex post-translational modifications
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Use fusion tags that enhance solubility (e.g., SUMO, thioredoxin)
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Optimize induction conditions (lower temperature, reduced inducer concentration)
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Include protease inhibitors throughout purification
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Carefully control protein concentration to prevent aggregation
How can researchers troubleshoot expression and purification issues with recombinant Danio rerio Collectin-12?
When troubleshooting expression and purification issues:
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Low expression levels:
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Optimize codon usage for the expression system
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Test different promoter systems
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Evaluate alternative host strains
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Consider expressing sub-domains separately if the full-length protein proves challenging
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Insoluble protein/inclusion bodies:
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Reduce induction temperature (e.g., 16-20°C)
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Decrease inducer concentration
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Use solubility-enhancing fusion partners
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Consider refolding protocols if inclusion bodies are unavoidable
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Proteolytic degradation:
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Use protease-deficient host strains
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Include protease inhibitor cocktails during purification
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Reduce purification time by optimizing protocols
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Maintain samples at 4°C throughout processing
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Poor purity after IMAC:
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Optimize imidazole concentration in wash buffers
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Consider a second purification step (ion exchange, size exclusion)
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Evaluate alternative affinity tags if His-tag performance is suboptimal
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Loss of activity after purification:
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Verify oligomerization status under non-reducing conditions
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Test different buffer compositions
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Add stabilizing agents (trehalose, glycerol)
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Minimize freeze-thaw cycles
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Systematic optimization of these parameters can significantly improve the yield and quality of recombinant Danio rerio Collectin-12.
What quality control measures should be implemented when working with recombinant Danio rerio Collectin-12?
Comprehensive quality control for recombinant Danio rerio Collectin-12 should include:
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Identity verification:
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SDS-PAGE under reducing and non-reducing conditions
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Western blot with anti-His antibody or specific anti-Collectin-12 antibodies
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Mass spectrometry peptide mapping
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N-terminal sequencing
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Purity assessment:
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Structural integrity:
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Circular dichroism spectroscopy to assess secondary structure
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Thermal shift assays to evaluate stability
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Native PAGE to assess oligomerization state
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Functional validation:
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Binding assays with known ligands
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Complement activation assays if applicable
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Cell-based functional assays relevant to research context
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Endotoxin testing:
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Limulus Amebocyte Lysate (LAL) assay to ensure preparations are endotoxin-free, particularly for cell culture applications
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Stability monitoring:
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Accelerated stability studies under different storage conditions
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Regular testing of long-term stored aliquots
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Implementation of these quality control measures ensures consistency between batches and reliable experimental results when using recombinant Danio rerio Collectin-12.
Can zebrafish models advance our understanding of Collectin-12 function in human health and disease?
Zebrafish models offer several advantages for studying Collectin-12 function relevant to human health:
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Developmental accessibility: The transparency and external development of zebrafish embryos allow real-time visualization of developmental processes involving Collectin-12, particularly in neural crest-derived tissues .
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Genetic manipulation: The ease of genetic manipulation in zebrafish through morpholinos, CRISPR/Cas9, and transgenic approaches facilitates functional studies of Collectin-12 in vivo.
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High-throughput screening: Zebrafish embryos can be used for small molecule screening to identify compounds that modulate Collectin-12 function, potentially identifying therapeutic leads.
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Conservation of developmental programs: The single-cell transcriptomic analysis of neural crest lineages in zebrafish has revealed evolutionarily conserved signatures, including Hox gene expression patterns that may interact with Collectin-12 function . This conservation increases the translational potential of zebrafish findings to human contexts.
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Disease modeling: Zebrafish models of diseases where Collectin-12 may play a role can provide insights into pathological mechanisms and potential therapeutic interventions.
The comprehensive cell type atlas of posterior sox10-expressing cells in zebrafish provides a valuable resource for investigating Collectin-12 function in specific developmental contexts with potential human disease relevance .
