Recombinant Danio rerio Protein kinase C-binding protein NELL2 (nell2), partial

What is Protein kinase C-binding protein NELL2 in Danio rerio?

Protein kinase C-binding protein NELL2, also known as NEL-like protein 2, is a protein in zebrafish (Danio rerio) that interacts with Protein kinase C (PKC), a multigene family of enzymes consisting of at least 11 isoforms involved in various signal transduction pathways . NELL2 is identified in the UniProt database with accession number A1A5Y0 and plays potential roles in cellular signaling pathways that regulate gene expression .

What are the optimal storage conditions for recombinant NELL2 protein?

Recombinant Danio rerio NELL2 protein should be stored at -20°C, while extended storage requires conservation at -20°C or -80°C . Repeated freezing and thawing is not recommended as it may impact protein stability and activity. For short-term usage, working aliquots can be maintained at 4°C for up to one week . These storage conditions help maintain the structural integrity and functional properties of the recombinant protein for experimental applications.

How should recombinant NELL2 protein be reconstituted for experimental use?

The recommended reconstitution protocol involves briefly centrifuging the vial prior to opening to bring contents to the bottom, then reconstituting the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage of reconstituted protein, addition of glycerol to a final concentration of 5-50% (with 50% being the standard default) is recommended before aliquoting and storing at -20°C/-80°C . This approach minimizes freeze-thaw cycles while maintaining protein stability.

What is the shelf life of recombinant NELL2 protein?

The shelf life depends on multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself . Generally, liquid preparations have a shelf life of approximately 6 months when stored at -20°C/-80°C, while lyophilized forms maintain stability for up to 12 months at -20°C/-80°C . Proper storage and handling protocols are essential for maintaining protein activity throughout the expected shelf life.

How can protein purity be verified when working with recombinant NELL2?

The recombinant NELL2 protein is typically supplied with >85% purity as verified by SDS-PAGE . Researchers should conduct their own verification of protein purity using SDS-PAGE, comparing band patterns with the expected molecular weight of NELL2. For more precise purity assessment, additional methods such as size exclusion chromatography or mass spectrometry might be employed. Western blotting using anti-NELL2 antibodies can confirm the identity of the protein while validating purity claims.

What methods can be used to study NELL2 interactions with Protein kinase C in zebrafish models?

To study NELL2-PKC interactions in zebrafish, researchers can employ co-immunoprecipitation assays using antibodies against either NELL2 or specific PKC isoforms of interest, followed by immunoblotting to detect the binding partner . Alternatively, yeast two-hybrid systems or fluorescence resonance energy transfer (FRET) techniques using fluorescently tagged NELL2 and PKC isoforms can reveal direct interactions. Transient-transfection assays in cell cultures derived from zebrafish can also be employed to assess functional consequences of these interactions, similar to studies conducted with PKC isoforms in mammalian systems .

How can NELL2 expression patterns be analyzed in zebrafish tissues?

NELL2 expression patterns can be analyzed using multiple complementary techniques. In situ hybridization can detect NELL2 mRNA in whole zebrafish embryos or tissue sections. Immunohistochemistry using anti-NELL2 antibodies can visualize protein localization within tissues. For quantitative assessment, qRT-PCR can measure NELL2 transcript levels across different tissues or developmental stages. Single-cell RNA sequencing approaches, similar to those used for other zebrafish cell types, can provide high-resolution expression data in specific cell populations .

What are the considerations for designing NELL2 knockdown or knockout experiments in zebrafish?

When designing NELL2 knockdown/knockout experiments, researchers should consider morpholino oligonucleotides for transient knockdown in embryos, or CRISPR-Cas9 for permanent gene editing. Target design should account for potential off-target effects and functional domains within the NELL2 protein. Validation strategies should include qRT-PCR or Western blotting to confirm reduced expression, and phenotypic characterization focused on systems where PKC signaling is important. As PKC isoforms play roles in multiple signaling pathways including those involved in gene expression regulation , researchers should examine potential developmental, neurological, and cell signaling phenotypes.

What is the potential role of NELL2 in PKC signaling pathways in zebrafish?

Based on its designation as a PKC-binding protein, NELL2 likely participates in PKC-mediated signal transduction in zebrafish. PKC family members, particularly PKC-alpha and PKC-epsilon, enhance activities of multiple signaling pathways including c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF cascades that converge on the serum response element (SRE) and regulate gene expression . NELL2 may function as a regulator or scaffold for specific PKC isoforms, potentially modulating their activation, substrate specificity, or subcellular localization in these pathways.

How might NELL2 function differ between zebrafish and other vertebrate models?

While direct comparative data is limited in the provided search results, functional analysis should consider evolutionary conservation and divergence. Researchers should examine sequence homology between zebrafish NELL2 and its orthologs in other vertebrates, particularly focusing on PKC-binding domains. Functional assays comparing binding affinities and downstream signaling effects across species can reveal evolutionary adaptations. Zebrafish-specific functions might be identified by examining expression patterns during development and in adult tissues compared to other model organisms, potentially revealing specialized roles in aquatic vertebrates.

Could NELL2 play a role in neuroepithelial cell function in zebrafish?

Neuroepithelial cells (NECs) in zebrafish gills function as respiratory chemoreceptors that detect changes in O₂ and CO₂/H⁺ levels . While direct evidence for NELL2 involvement in NEC function is not presented in the search results, the protein's signaling capabilities through PKC interaction make it a candidate for investigation. Researchers could examine NELL2 expression in NECs using single-cell RNA sequencing approaches similar to those described for identifying other markers in these cells . Functional studies could assess whether NELL2 modulates NEC responses to hypoxia or other environmental changes.

How can researchers address inconsistent results when working with recombinant NELL2 protein?

Inconsistent results may stem from several factors. First, verify protein quality by assessing activity immediately after reconstitution versus after storage. Consider batch-to-batch variation by requesting certificate of analysis data and conducting standardization experiments. Experimental conditions including buffer composition, pH, temperature, and presence of cofactors may significantly impact NELL2 function. Additionally, ensure that any interacting proteins (particularly PKC isoforms) are present in appropriate concentrations and activation states when studying functional interactions .

What controls should be included when studying NELL2-PKC interactions?

Appropriate controls for studying NELL2-PKC interactions include positive controls using known PKC-interacting proteins and negative controls with proteins not expected to interact with PKC. When examining specific PKC isoforms, dominant-negative and constitutively active mutants of different PKC isoforms (similar to those used in PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta studies ) can help determine isoform specificity. Additional controls should address potential non-specific binding by using truncated versions of NELL2 lacking the PKC-binding domain.

How can researchers distinguish between direct and indirect effects of NELL2 on cellular processes?

To distinguish direct from indirect NELL2 effects, researchers should employ time-course experiments to establish temporal relationships between NELL2 activity and downstream events. Direct protein-protein interactions can be confirmed using in vitro binding assays with purified components. Mutational analysis targeting specific binding domains within NELL2 can determine which interactions are essential for observed effects. Pharmacological approaches using specific inhibitors of PKC isoforms or downstream effectors can help delineate the signaling pathway and identify where NELL2 exerts its effects within the cascade .

What are potential explanations for discrepancies between in vitro and in vivo findings regarding NELL2 function?

Discrepancies between in vitro and in vivo findings may result from differences in protein concentration, post-translational modifications, or the absence of essential cofactors or interaction partners in simplified in vitro systems. The cellular microenvironment in vivo, including compartmentalization of signaling components, may significantly affect NELL2 function. Additionally, compensatory mechanisms that are activated in vivo following genetic manipulation may mask phenotypes that would be apparent in isolated systems. Researchers should employ multiple complementary approaches, combining biochemical assays, cell culture models, and whole-organism studies to develop a comprehensive understanding of NELL2 biology.

How might high-throughput technologies advance our understanding of NELL2 function in zebrafish?

High-throughput approaches can significantly enhance our understanding of NELL2 biology. Proteomics techniques such as affinity purification coupled with mass spectrometry can identify the complete interactome of NELL2 beyond PKC isoforms. CRISPR screens targeting potential interaction partners could reveal functional relationships. Single-cell RNA sequencing, similar to that employed for zebrafish gill cells , can provide high-resolution data on cell types expressing NELL2 and how its expression changes under various physiological conditions or developmental stages. These approaches would generate comprehensive datasets that could reveal previously unrecognized functions of NELL2.

What are potential translational applications of zebrafish NELL2 research to human health?

Research on zebrafish NELL2 could have translational relevance to human health through comparative studies with human NELL2. If zebrafish NELL2 functions in PKC signaling pathways similar to those in humans, the zebrafish model could provide insights into pathologies associated with dysregulated PKC signaling, such as cancer, neurological disorders, or inflammatory conditions . The optical transparency and genetic tractability of zebrafish make them valuable for high-throughput drug screening to identify compounds that modulate NELL2-PKC interactions, potentially leading to novel therapeutic approaches for human diseases involving these pathways.

How might environmental factors influence NELL2 expression and function in zebrafish?

Environmental factors such as hypoxia might influence NELL2 expression and function in zebrafish. The search results indicate that hypoxia exposure induces transcriptomic changes in neuroepithelial cells of zebrafish gills, with affected cells showing high expression of genes involved in cell cycle control and proliferation . Future research could investigate whether NELL2 expression is similarly modulated by hypoxia or other environmental stressors, and whether such changes affect PKC signaling pathways. This research direction has ecological relevance and could reveal how evolutionary adaptations in signaling pathways help organisms respond to environmental challenges.