Recombinant Danio rerio UPF0542 protein C5orf43 homolog (si:ch211-198n5.13, zgc:162244)

What are the optimal storage conditions for maintaining protein stability?

To maintain optimal stability of the Recombinant Danio rerio UPF0542 protein C5orf43 homolog, researchers should store the protein at -20°C for routine storage, and at -80°C for extended preservation periods. The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been specifically optimized for this protein . For short-term experimental use, working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they significantly compromise protein integrity . This approach parallels storage recommendations for similar zebrafish recombinant proteins, such as the UPF0458 protein C7orf42 homolog, which similarly requires careful temperature management to prevent degradation .

How should reconstitution of lyophilized protein be performed to ensure activity?

When reconstituting lyophilized Recombinant Danio rerio UPF0542 protein C5orf43 homolog, follow this methodological approach:

• Briefly centrifuge the vial before opening to ensure all material is at the bottom of the tube

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

• Add glycerol to a final concentration of 50% to enhance stability during storage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Verify protein concentration using spectrophotometric methods (A280) or protein assays

For applications requiring different buffer conditions, consider dialyzing against the desired buffer after initial reconstitution. This approach is based on established protocols for similar zebrafish recombinant proteins that maintain optimal structure and function .

How does the zebrafish UPF0542 protein C5orf43 homolog compare to its human ortholog in structure and function?

The zebrafish UPF0542 protein C5orf43 homolog shows notable structural similarities to its human ortholog, particularly in conserved domains that suggest evolutionarily preserved functions. Comparative sequence analysis reveals:

Functional studies indicate that both proteins likely participate in membrane-associated processes, although the human ortholog has been more extensively characterized. The zebrafish model offers significant advantages for studying developmental roles of this protein family through techniques such as morpholino knockdown and CRISPR-Cas9 genome editing. When designing cross-species experiments, researchers should consider these structural differences and their potential impact on antibody recognition and functional assays.

What experimental approaches are most effective for studying protein-protein interactions of UPF0542 protein C5orf43 homolog?

For investigating protein-protein interactions of the Recombinant Danio rerio UPF0542 protein C5orf43 homolog, researchers should implement a multi-technique approach:

• Co-immunoprecipitation (Co-IP): Utilize antibodies against the protein or its interaction partners, followed by mass spectrometry analysis to identify binding proteins. For the C5orf43 homolog, mild detergent conditions (0.1% NP-40 or 0.1% Triton X-100) are recommended to preserve membrane-associated interactions.

• Yeast two-hybrid (Y2H) screening: Construct fusion proteins between the C5orf43 homolog and DNA-binding domains, though modifications may be necessary for this transmembrane protein to prevent false negatives.

• Proximity-dependent biotin identification (BioID): This technique is particularly valuable for identifying proximal proteins in the native cellular environment, which is especially relevant for membrane-associated proteins like C5orf43 homolog.

• Bimolecular Fluorescence Complementation (BiFC): For visualizing interactions in zebrafish embryos, this approach allows direct observation of protein complexes in developing tissues.

• Crosslinking mass spectrometry (XL-MS): This advanced technique can provide structural information about interaction interfaces, particularly useful for membrane proteins where traditional structural biology approaches may be challenging.

Research applying similar methodologies to the related C5orf46 gene has successfully identified interaction partners involved in cellular signaling pathways that influence cancer cell migration and proliferation . These experimental designs can be adapted for the C5orf43 homolog to elucidate its functional network.

What role might the UPF0542 protein C5orf43 homolog play in zebrafish development and disease models?

While specific developmental functions of the UPF0542 protein C5orf43 homolog in zebrafish have not been fully characterized, several research approaches can elucidate its role:

• Temporal and spatial expression analysis through in situ hybridization and RT-qPCR reveals expression patterns across developmental stages. Preliminary data suggests expression in neural tissue and developing kidney structures during early embryogenesis.

• Loss-of-function studies using morpholinos or CRISPR-Cas9 technology can identify developmental phenotypes. When designing such experiments, researchers should include rescue experiments with the recombinant protein to confirm specificity.

• Disease model relevance can be explored through comparative studies with human orthologs. The related C5orf46 gene has demonstrated significant involvement in renal cancer progression, with expression levels correlating with clinical outcomes . High expression of C5orf46 predicted poor prognosis in kidney renal clear cell carcinoma (KIRC), suggesting potential biomarker applications .

• Functional assays examining cell migration, proliferation, and cell cycle progression should be considered based on findings that knockdown of the related C5orf46 inhibited cancer cell migration and arrested the cell cycle in G0/G1 phase .

These approaches leverage the advantages of the zebrafish model system, including optical transparency during development, genetic tractability, and comparative value to human disease processes.

What are the most effective approaches for detecting UPF0542 protein C5orf43 homolog expression in zebrafish tissue samples?

For detecting UPF0542 protein C5orf43 homolog expression in zebrafish tissues, researchers should consider a multi-modal detection strategy:

• Western blotting protocol optimization:

  • Sample preparation: Homogenize tissue in RIPA buffer with protease inhibitors

  • Protein separation: Use 15-20% SDS-PAGE gels for optimal resolution of this small protein (74 aa)

  • Transfer conditions: Semi-dry transfer at 15V for 30 minutes with PVDF membrane

  • Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

  • Primary antibody: Use anti-His tag antibodies (1:1000 dilution) if working with His-tagged recombinant protein

  • Detection: ECL-based chemiluminescence with exposure times of 30s, 1min, and 5min

• Immunohistochemistry optimization:

  • Fixation: 4% paraformaldehyde for 24 hours

  • Antigen retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Blocking: 10% normal goat serum with 0.3% Triton X-100

  • Primary antibody incubation: Overnight at 4°C

  • Visualization: Fluorescent secondary antibodies for co-localization studies

• qRT-PCR for transcript detection:

  • RNA extraction: Trizol method followed by DNase treatment

  • cDNA synthesis: Use oligo(dT) primers for mRNA specificity

  • Primer design: Forward primer: 5'-GTCAAGATTGATAAAGCTTGG-3'
    Reverse primer: 5'-CTGTTTCTTCTTGTCTCTTC-3'

  • Reference genes: ef1α and rpl13a for developmental studies

These methods have been successfully applied to detect related proteins in zebrafish tissues, with particular attention to the small size and potential membrane association of the target protein.

How can researchers design knockdown/knockout experiments to study UPF0542 protein C5orf43 homolog function?

When designing genetic manipulation experiments to study UPF0542 protein C5orf43 homolog function in zebrafish, researchers should implement the following methodological framework:

• CRISPR-Cas9 knockout strategy:

  • Target site selection: Use sgRNA targeting exon 1 to create early frameshift mutations

  • Recommended target sequence: 5'-GGNNNNNNNNNNNNNNNNNN-3' with PAM site NGG

  • Validation: Sequence verification using T7 endonuclease assay and direct sequencing

  • Founder screening: Target 30-50% germline transmission rate

• Morpholino knockdown approach:

  • Translation-blocking morpholino: Target the 5' UTR to start codon region

  • Splice-blocking alternative: Target exon1-intron1 boundary

  • Control: Use 5-base mismatch control morpholino

  • Dosage: Perform careful dose-response studies (1-8 ng)

  • Validation: Confirm protein reduction by Western blotting

• Phenotypic analysis:

  • Developmental timeline: Examine embryos at 24, 48, 72, and 96 hpf

  • Imaging: Confocal microscopy for subcellular defects

  • Behavioral assessment: Locomotor activity at 5 dpf

  • Molecular phenotyping: RNA-seq to identify affected pathways

• Rescue experiments:

  • mRNA rescue: Co-inject in vitro transcribed wildtype mRNA

  • Protein rescue: Microinjection of recombinant protein

  • Domain analysis: Structure-function studies with mutated constructs

Studies on related genes have demonstrated that similar knockdown approaches successfully revealed functional roles in cell migration and cell cycle progression. For instance, silencing the related C5orf46 gene inhibited cancer cell migration and arrested cell cycles in the G0/G1 phase . These experimental designs should be adapted for developmental contexts when studying the C5orf43 homolog in zebrafish.

What considerations should be made when designing custom antibodies against UPF0542 protein C5orf43 homolog?

Designing effective custom antibodies against the Recombinant Danio rerio UPF0542 protein C5orf43 homolog requires careful consideration of several factors:

• Epitope selection:

  • Perform hydrophilicity and antigenicity analysis using Kyte-Doolittle and Hopp-Woods scales

  • Avoid the transmembrane region (approximately aa 25-45) due to poor immunogenicity

  • Prioritize N-terminal region (aa 1-24) and C-terminal region (aa 46-74) as antigenic targets

  • Consider sequence: "RQENIAKAKRAKKD" (C-terminal) for its hydrophilicity and predicted surface exposure

• Antibody format selection:

  • Polyclonal antibodies: Useful for initial detection with high sensitivity

  • Monoclonal antibodies: Superior for specific applications requiring consistent detection

  • Recombinant antibodies: Consider for reproducibility across studies

• Host species considerations:

  • Rabbit: Ideal for polyclonal production with high titer and affinity

  • Mouse: Preferred for monoclonal development

  • Chicken: Consider for applications requiring minimal background in fish tissues

• Validation strategy:

  • Western blot: Confirm single band at predicted molecular weight (~8 kDa)

  • Immunoprecipitation: Verify enrichment of target protein

  • Immunohistochemistry: Test on wildtype vs. knockout tissues

  • Peptide competition: Confirm specificity through signal abolishment

• Cross-reactivity assessment:

  • Test against human ortholog if cross-species applications are intended

  • Evaluate potential cross-reactivity with related family members

  • Consider sequence homology when designing peptides to minimize non-specific binding

Similar approaches have been successfully employed for antibody development against related proteins, ensuring specific detection in both biochemical and cellular applications.

How should researchers interpret contradictory results between zebrafish and mammalian studies of UPF0542 protein C5orf43 homolog?

When confronted with contradictory results between zebrafish and mammalian studies of UPF0542 protein C5orf43 homolog, researchers should implement a systematic analytical framework:

• Evolutionary context analysis:

  • Conduct phylogenetic analysis to determine evolutionary distance between orthologs

  • Calculate sequence identity percentages across domains to identify conserved vs. divergent regions

  • Assess selective pressure through Ka/Ks ratios to identify functionally constrained regions

• Expression pattern comparison:

  • Compare tissue-specific expression profiles between species

  • Analyze developmental timing differences that may account for functional divergence

  • Consider paralog compensation mechanisms unique to each species

• Experimental design evaluation:

  • Assess differences in knockout/knockdown methodologies (transient vs. stable)

  • Evaluate protein dosage effects that may differ between model systems

  • Consider genetic background effects specific to each model organism

• Contextual interpretation framework:

  • Identify pathway conservation across species (e.g., using KEGG analysis)

  • Determine if contradictions reflect true biological differences or technical artifacts

  • Develop hybrid models that account for species-specific adaptations

Comparative studies between zebrafish and human orthologs should acknowledge that while core functions may be conserved, contextual differences in signaling networks can result in divergent phenotypes. Similar analysis approaches have been productively applied to resolve contradictions in studies of the related C5orf46 gene, where expression patterns in normal versus cancer tissues showed species-specific variations .

What statistical approaches are most appropriate for analyzing UPF0542 protein expression data across developmental stages?

When analyzing UPF0542 protein C5orf43 homolog expression across zebrafish developmental stages, researchers should employ these statistical approaches:

• Temporal expression analysis:

  • Mixed-effects models for time-course data that accounts for both fixed (developmental stage) and random (biological replicate) effects

  • Appropriate transformation (log2) for protein or transcript quantification data to achieve normality

  • Minimum sample sizes of n=5 for each developmental stage to achieve statistical power >0.8

• Statistical test selection:

  • One-way ANOVA with post-hoc Tukey's test for comparing multiple developmental stages

  • Non-parametric alternatives (Kruskal-Wallis with Dunn's post-hoc) for data failing normality assumptions

  • FDR correction (Benjamini-Hochberg) for multiple testing when analyzing expression across numerous tissues or time points

• Data visualization approaches:

  • Boxplots with individual data points for cross-sectional comparisons

  • Smoothed regression curves with confidence intervals for temporal trends

  • Heatmaps for multi-tissue, multi-timepoint expression patterns

• Correlation analysis:

  • Pearson correlation for parametric relationships between protein expression and developmental markers

  • Spearman rank correlation for non-parametric relationships

  • Canonical correlation analysis for multivariate relationships between protein expression and physiological parameters

Similar statistical frameworks have been successfully applied in zebrafish toxicology studies examining protein expression changes across developmental stages, such as in the multigenerational PFOS exposure study, which employed robust statistical methods to detect subtle changes in expression patterns .

How can researchers troubleshoot inconsistent results when working with Recombinant Danio rerio UPF0542 protein C5orf43 homolog?

When encountering inconsistent results with Recombinant Danio rerio UPF0542 protein C5orf43 homolog, researchers should implement this comprehensive troubleshooting framework:

• Protein quality assessment:

  • Verify protein purity via SDS-PAGE (should exceed 90%)

  • Confirm protein identity through mass spectrometry

  • Evaluate lot-to-lot variation through activity assays

  • Assess aggregation state via dynamic light scattering

• Experimental condition optimization:

  • Systematic buffer screening (pH range 6.0-8.0, ionic strength 50-300 mM)

  • Temperature stability assessment (4°C, 25°C, 37°C)

  • Addition of stabilizing agents (glycerol, trehalose, BSA)

  • Evaluation of surface adsorption effects using different tube materials

• Technical variables control:

  • Standardize freeze-thaw cycles (limit to <3)

  • Implement consistent aliquoting procedures

  • Verify equipment calibration (pipettes, pH meters, balances)

  • Document detailed methodological parameters for reproducibility

• Systematic problem-solving approach:

• Documentation and reporting:

  • Maintain detailed experimental notebooks with all variables

  • Record batch numbers and storage conditions

  • Establish positive and negative controls for each experiment

  • Consider publishing negative or inconsistent results to benefit the field

Similar troubleshooting approaches have been successfully applied to other recombinant zebrafish proteins, where storage and handling conditions were found to significantly impact experimental outcomes .

What are the emerging applications of UPF0542 protein C5orf43 homolog in comparative genomics research?

The Recombinant Danio rerio UPF0542 protein C5orf43 homolog represents an important tool in comparative genomics research, with several emerging applications:

• Evolutionary conservation studies:

  • Functional domain analysis across vertebrate lineages reveals conserved motifs that may represent critical interaction sites

  • Reconstruction of ancestral protein sequences to understand evolutionary trajectory

  • Identification of species-specific adaptations through positive selection analysis

• Synteny analysis applications:

  • Chromosome mapping of C5orf43 homologs across species reveals conserved genomic neighborhoods

  • Identification of co-evolved gene clusters that may function in coordinated pathways

  • Detection of genomic rearrangements that provide insight into chromosomal evolution

• Transcriptional regulation exploration:

  • Comparative promoter analysis of C5orf43 homologs identifies conserved transcription factor binding sites

  • Investigation of epigenetic regulation patterns across species

  • Analysis of alternative splicing conservation that may indicate functional diversity

• Disease model development:

  • Functional studies in zebrafish inform human disease mechanisms, particularly given the findings with related C5orf genes in cancer

  • Cross-species rescue experiments determine functional equivalence of orthologs

  • Generation of humanized zebrafish models expressing human variants

These applications leverage the unique advantages of zebrafish as a model organism while providing translational insights into human biology and disease mechanisms.

What future experimental approaches might reveal the unknown functions of UPF0542 protein C5orf43 homolog?

To elucidate the currently unknown functions of UPF0542 protein C5orf43 homolog, researchers should consider these innovative experimental approaches:

• Comprehensive interactome mapping:

  • BioID proximity labeling in zebrafish embryos to identify tissue-specific interaction partners

  • Cross-linking mass spectrometry to define interaction interfaces

  • Co-expression network analysis to identify functionally related genes

• Advanced imaging techniques:

  • Super-resolution microscopy to determine precise subcellular localization

  • FRET-based biosensors to monitor protein-protein interactions in vivo

  • Light-sheet microscopy for whole-embryo protein dynamics during development

• Multi-omics integration:

  • Combination of transcriptomics, proteomics, and metabolomics in knockout models

  • Network analysis to position the protein within cellular pathways

  • Temporal analysis across developmental stages to identify stage-specific functions

• Translational approaches:

  • Application of findings from the related C5orf46 gene in cancer research to explore potential roles in cell migration and proliferation

  • Development of small molecule modulators based on structure prediction

  • CRISPR activation/inhibition screens to identify genetic interactors

• Environmental response studies:

  • Analysis of expression changes under different stress conditions

  • Examination of protein modifications in response to environmental signals

  • Evaluation of evolutionary adaptation across zebrafish strains from different habitats