C3orf14 Antibody

What is the molecular structure and cellular localization of C3orf14?

C3orf14 (Chromosome 3 open reading frame 14) encodes a 15007.84 kD protein known as HT021, which is characterized by alpha helices spanning most of its length. The protein has a pre-modification isoelectric point of 5.57 and contains four potential phosphorylation sites, with at least two of these sites conserved across all orthologs. Structurally, C3orf14 is predicted to function as a DNA-binding protein and may assume a tertiary coiled-coil structure. While initially thought to localize to the nucleus, recent advanced imaging has revealed that GFP-tagged C3orf14 localizes closely to NIN (Ninein), suggesting it functions as a sub-distal centriolar appendage protein .

What is the evolutionary significance of C3orf14?

C3orf14 demonstrates remarkable evolutionary conservation, with orthologs identified across most major animal groups including mammals, monotremes, birds, reptiles, fish, and invertebrates. The gene traces back approximately 1000 million years to sea anemones, suggesting fundamental biological importance. The amino acid structure is highly conserved among mammals, while the secondary and tertiary structures remain highly conserved across all orthologs. This extraordinary conservation suggests C3orf14 performs a critical cellular function, despite being uncharacterized until recently. Notably, no orthologs have been identified in plants or bacteria, indicating a potentially animal-specific role .

What are the genomic characteristics of the C3orf14 gene?

The C3orf14 gene is located at chromosome 3p14.2 near the fragile site FRBA3, which falls between this gene and the centromere. The gene is composed of 6 exons that encode the HT021 protein. C3orf14 is also known by several aliases including LOC57415, FLJ94553, and FLJ17473. Gene orthologs in other organisms are typically identified as c3orf14-like, though some are designated as LOC57415-like or HT021-like (referring to the protein name). In human genomic databases, C3orf14 is indexed under accession number Q9HBI5 (UniProt) and is referenced under HGNC:25024 and RefSeq NM_020685.3 .

How do researchers validate C3orf14 antibody specificity for experimental applications?

Validating antibody specificity for C3orf14 requires multiple complementary approaches due to its relatively uncharacterized nature. Researchers typically begin with Western blot analysis to confirm the antibody detects a protein of the expected molecular weight (approximately 15 kDa). This should be accompanied by knockout/knockdown controls where the target protein is depleted, resulting in reduced or absent signal. For immunocytochemistry applications, co-localization experiments with established centrosomal markers (particularly distal appendage proteins like CEP89 or Ninein) provide spatial validation. Additionally, researchers can validate specificity by transiently expressing tagged C3orf14 and confirming antibody co-localization with the expressed protein. For polyclonal antibodies particularly, pre-absorption with the immunizing antigen (Recombinant Human Uncharacterized protein C3orf14, amino acids 9-128) should abolish specific staining .

What is the role of C3orf14 in centrosome biology based on current research?

Recent proximity mapping studies have identified C3orf14 as a previously uncharacterized component of the centrosome-cilium interface. Specifically, BioID proximity labeling revealed that C3orf14 interacts with the distal appendage protein CEP89, and this interaction was confirmed by reciprocal experiments using C3orf14 as a bait protein. Advanced imaging using GFP-tagged C3orf14 demonstrated localization closely resembling Ninein, suggesting C3orf14 is a sub-distal centriolar appendage protein. Furthermore, functional studies have implicated C3orf14 in centriole amplification pathways, as it was identified among factors whose knockdown suppressed this process. This emerging evidence positions C3orf14 as a potentially important structural or regulatory component of centriolar appendages, which are crucial for microtubule anchoring and cilium formation .

What protein-protein interaction networks involve C3orf14?

Comprehensive protein interaction mapping using BioID proximity labeling has uncovered that C3orf14 exists within a specific interaction network at the centrosome. Most notably, C3orf14 shows strong interaction with CEP89, a known distal appendage protein. When C3orf14 itself was used as a BioID bait protein, researchers confirmed this reciprocal interaction along with nine additional shared proximity interactions. C3orf14's localization pattern and interaction profile place it within the sub-distal centriolar appendage subnetwork, which includes other proteins such as CEP19 and CEP128. These interactions position C3orf14 within the broader centrosome-cilium interaction landscape, suggesting potential roles in centrosomal protein organization, centriole duplication, or cilium formation, though the precise functional significance of these interactions remains to be fully elucidated .

What are the optimal protocols for using C3orf14 antibodies in Western blot applications?

For Western blot applications with C3orf14 polyclonal antibodies, researchers should optimize several parameters specific to this relatively understudied protein. Sample preparation should include phosphatase inhibitors to preserve potentially important phosphorylation states at the four identified sites. Proteins should be separated on 12-15% polyacrylamide gels to ensure optimal resolution of the approximately 15 kDa target. After transfer to PVDF or nitrocellulose membranes, blocking with 5% BSA in TBST is recommended to minimize background. Primary antibody dilutions of 1:500-1:1000 are typically effective with overnight incubation at 4°C. Given the tissue distribution pattern, lysates from brain, testis, or kidney tissues often provide positive controls, while using cell lines with CRISPR-mediated C3orf14 knockout serves as an essential negative control. Importantly, researchers should be aware that post-translational modifications may affect migration patterns, potentially resulting in detection at molecular weights differing from the predicted 15 kDa .

What considerations are important for immunohistochemistry applications with C3orf14 antibodies?

For immunohistochemistry applications with C3orf14 antibodies, researchers should implement specific optimization strategies. Effective antigen retrieval is critical - heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally recommended for paraffin-embedded sections. Due to the nuclear and centrosomal localization patterns of C3orf14, permeabilization must be carefully optimized with 0.1-0.3% Triton X-100 to allow antibody access without disrupting fine centrosomal structures. Primary antibody dilutions typically range from 1:100-1:200 with overnight incubation at 4°C. For fluorescent detection, secondary antibodies with minimal cross-reactivity to other species are essential for clean signal detection. Co-staining with established centrosomal markers (γ-tubulin for general centrosome detection or CEP89 for distal appendages) provides important localization context. Confocal or super-resolution microscopy is strongly recommended to accurately visualize the sub-distal appendage localization pattern of C3orf14, as conventional fluorescence microscopy lacks the resolution to distinguish these fine structures .

What are the recommended approaches for using C3orf14 antibodies in ELISA applications?

When implementing ELISA protocols with C3orf14 antibodies, researchers should consider the protein's structural characteristics. For sandwich ELISA development, using an antibody pair targeting different epitopes is essential - typically combining the polyclonal antibody raised against amino acids 9-128 with a monoclonal antibody targeting a different region. Standard curves should be generated using recombinant C3orf14 protein at concentrations ranging from 0.1-1000 ng/mL. Sample dilution optimization is critical, as C3orf14 concentration varies significantly between tissue types. Background reduction requires careful blocking with 1-2% BSA in PBS and thorough washing steps. For competitive ELISA formats, pre-incubation of the antibody with varying concentrations of the antigen creates a standard displacement curve. Validation across multiple sample types is necessary, as C3orf14 expression patterns differ between tissues, with typically higher levels observed in brain and testis compared to other tissues .

How can researchers effectively use C3orf14 antibodies for studying centrosome-cilium interactions?

To study centrosome-cilium interactions using C3orf14 antibodies, researchers should implement a multi-faceted approach. First, super-resolution microscopy techniques (3D-SIM, STED, or STORM) are essential for precise localization, as conventional microscopy cannot resolve the sub-distal appendage structures where C3orf14 resides. Co-immunostaining with multiple centrosomal markers is crucial: pair C3orf14 antibodies with CEP89 (confirmed interactor), Ninein (similar localization pattern), and additional markers like γ-tubulin (centrosome), CEP164 (distal appendages), and acetylated tubulin (ciliary axoneme). For functional studies, researchers should consider proximity labeling methods like BioID or TurboID with C3orf14 as the bait to identify additional interaction partners beyond the established CEP89 connection. CRISPR-Cas9 knockout or depletion of C3orf14 followed by detailed phenotypic analysis of centrosome structure, ciliogenesis efficiency, and cilium morphology will provide insights into its functional role. Time-resolved imaging during cell cycle progression and ciliogenesis can reveal dynamic aspects of C3orf14 localization and function not captured in static analyses .

What are the critical controls needed when investigating C3orf14 protein interactions?

When investigating C3orf14 protein interactions, rigorous controls are essential due to its location in a complex cellular structure. For co-immunoprecipitation experiments, both overexpressed tagged controls and endogenous protein precipitation should be performed. Negative controls must include IgG from the same species as the primary antibody and precipitations from cells where C3orf14 has been depleted via CRISPR-Cas9 or RNAi. For proximity labeling studies (BioID/TurboID), controls should include a non-targeting BirA fusion (e.g., BirA-GFP) expressed at comparable levels and localized to similar cellular compartments to identify nonspecific biotinylation. Researchers should validate novel interactions through reciprocal experiments (using the putative interactor as bait) as demonstrated in the identification of the C3orf14-CEP89 interaction. Additionally, fluorescence microscopy correlation analyzing co-localization patterns between C3orf14 and candidate interactors provides spatial validation to complement biochemical approaches. For centrosomal proteins specifically, cell cycle-dependent changes in interactions should be monitored, as appendage proteins undergo significant reorganization during centrosome duplication and maturation .

What troubleshooting approaches should be considered when C3orf14 antibodies show inconsistent results?

When encountering inconsistent results with C3orf14 antibodies, researchers should systematically evaluate several variables. First, epitope accessibility may be compromised due to C3orf14's localization at the centrosomal appendages; alternative fixation methods should be tested, comparing paraformaldehyde, methanol, and glutaraldehyde fixation, which differentially preserve epitopes and centrosome structure. Second, phosphorylation state sensitivity should be assessed, as C3orf14 contains four potential phosphorylation sites that may affect antibody recognition. Researchers can treat samples with phosphatase inhibitors or lambda phosphatase to determine if phosphorylation influences detection. Third, expression level variability across cell types and cell cycle stages may explain inconsistent detection; synchronization protocols can help determine if C3orf14 expression or localization is cell cycle-dependent. For Western blot applications specifically, optimizing extraction conditions is critical, as centrosomal proteins often require specialized lysis buffers containing detergents like NP-40 or Triton X-100 at appropriate concentrations to effectively solubilize centrosome-associated proteins while maintaining antibody epitopes .

What is known about C3orf14 in relation to ciliopathies and microcephalies?

Although direct causative mutations in C3orf14 have not yet been definitively linked to specific human diseases, its centrosomal localization and interaction network place it within pathways relevant to ciliopathies and microcephalies. C3orf14 interacts with the distal appendage protein CEP89 and exhibits similar localization to Ninein, positioning it among proteins crucial for proper centrosome function. The comprehensive protein interaction study identified 55 polypeptides linked to ciliopathies or microcephalies within the centrosome interaction network that includes C3orf14. Additionally, the centriole amplification screen that identified C3orf14 as a hit also flagged WDR62, a known microcephaly gene. The extraordinary evolutionary conservation of C3orf14 across animal lineages further suggests its disruption could have significant developmental consequences. Researchers investigating unexplained ciliopathies or microcephalies should consider C3orf14 as a candidate for sequencing and functional studies, particularly in cases showing centrosome abnormalities without mutations in known disease genes .

How can researchers effectively design experiments to investigate C3orf14 function in model organisms?

Designing effective experiments to study C3orf14 function in model organisms requires strategic approaches that account for its evolutionary conservation. For mammalian models, CRISPR-Cas9 genome editing should target conserved regions of C3orf14, particularly the phosphorylation sites and alpha helices preserved across species. Phenotypic analysis should focus on centrosome structure, cilia formation, cell division abnormalities, and developmental outcomes, with particular attention to tissues where centrosome dysfunction typically manifests (brain, kidney, retina). For cellular models, inducible knockdown or knockout systems allow temporal control to distinguish between immediate consequences and compensatory responses. Since C3orf14 orthologs exist in zebrafish and potentially Drosophila models, comparative studies across species can reveal evolutionarily conserved functions. Rescue experiments introducing wild-type or mutant C3orf14 into knockout backgrounds provide particularly valuable functional insights. For all models, researchers should implement quantitative phenotyping methods, including automated high-content imaging of centrosome number, position, and morphology, as well as measurements of cilia length, frequency, and function .

What analytical approaches are recommended for interpreting C3orf14 expression data in tissue samples?

When analyzing C3orf14 expression data across tissue samples, researchers should implement several specialized analytical approaches. Single-cell RNA sequencing analysis is particularly valuable, as C3orf14 may show cell type-specific expression patterns that bulk tissue analysis could obscure. Correlation analysis with known centrosomal, cell cycle, and ciliary genes can reveal functional associations and potential regulatory networks. Since C3orf14 is evolutionarily conserved, cross-species expression comparison can highlight tissues where its function is most critical. Developmental time-course analysis is essential, as centrosomal gene expression often varies during development, particularly in neurogenesis. For immunohistochemical studies of tissue sections, quantitative image analysis should measure both expression levels and subcellular localization patterns, ideally using machine learning algorithms trained to recognize centrosomal structures. When examining potential disease associations, stratification by genotype (particularly for genes encoding known C3orf14 interactors like CEP89) may reveal context-dependent expression patterns. Finally, integrated multi-omics approaches combining transcriptomic, proteomic, and phosphoproteomic data provide the most comprehensive view of C3orf14 regulation and function across tissues .