CCDC113 Antibody

What is CCDC113 and why is it important in cellular research?

CCDC113 is a 377-amino acid protein that functions as a component of centriolar satellites and contributes to primary cilium formation. It has subcellular localization primarily in the cytoplasm and exists in up to two different isoforms in humans . Recent research has identified CCDC113's potential roles in ciliary assembly and function, making it an important target for studies related to ciliopathies and cell division . Understanding this protein is particularly relevant for researchers investigating cellular structures and their relation to disease states, as altered expression has been associated with various pathological conditions including cancer .

Which detection methods are most effective for CCDC113 localization in cellular studies?

For subcellular localization studies, immunofluorescence techniques using validated anti-CCDC113 antibodies provide the most reliable results. Confocal microscopy following immunofluorescence staining has successfully demonstrated CCDC113's predominant cytoplasmic localization in colorectal cancer cells and other cell types . When designing these experiments, it's essential to use appropriate controls and cell fixation protocols that preserve cytoplasmic structures. For co-localization studies with centriolar markers, super-resolution microscopy may provide additional insights into the protein's precise localization within centriolar satellites .

What are the key considerations when selecting a CCDC113 antibody for research?

When selecting a CCDC113 antibody, researchers should consider:

• Target species specificity: Ensure the antibody recognizes your species of interest (human, mouse, rat, etc.)

• Application compatibility: Verify validation for your specific application (Western blot, IHC, ICC/IF, ELISA)

• Epitope location: Some antibodies target specific regions (e.g., C-terminal) which may affect detection of different isoforms

• Validation data: Review available literature citations and validation images

• Format and conjugation: Consider whether unconjugated or conjugated formats (FITC, etc.) are needed

CCDC113 orthologs have been reported in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, so careful selection based on experimental needs is critical .

What are the optimal conditions for using CCDC113 antibodies in Western blot applications?

For optimal Western blot detection of CCDC113:

• Sample preparation: Use RIPA or similar buffers with protease inhibitors

• Protein loading: 20-40 μg of total protein per lane is typically sufficient

• Gel percentage: 10-12% SDS-PAGE gels are appropriate for the 44.2 kDa CCDC113 protein

• Transfer conditions: Semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 100V for 1 hour

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

• Primary antibody dilution: Typically 1:500-1:2000 depending on antibody quality (check manufacturer recommendations)

• Incubation: Overnight at 4°C with gentle agitation

• Detection: HRP-conjugated secondary antibodies with ECL detection systems

When interpreting results, be aware that CCDC113 may show bands at approximately 44 kDa (canonical form) with possible additional bands representing different isoforms .

How should researchers design knockdown experiments to study CCDC113 function?

When designing CCDC113 knockdown studies:

• Select appropriate targeting strategy:

  • shRNA approach: Design 2-3 different shRNAs targeting different regions of CCDC113 mRNA

  • siRNA approach: Pool of 3-4 siRNAs often provides more consistent knockdown

  • CRISPR-Cas9: For complete knockout studies

• Validation of knockdown efficiency:

  • qRT-PCR to confirm mRNA reduction (as demonstrated in CRC studies)

  • Western blot to verify protein reduction

  • Include appropriate controls (scrambled/non-targeting sequences)

• Functional assays based on research question:

  • For proliferation: CCK-8 assays, colony formation assays

  • For migration: Wound-healing assays, transwell migration assays

  • For apoptosis: Flow cytometry with Annexin V/PI staining

  • For ciliary function: Immunofluorescence microscopy of ciliary markers

In published research, CCDC113 knockdown significantly inhibited the viability and proliferation of colorectal cancer cells while increasing apoptotic rates .

What methodological approaches are recommended for studying CCDC113 in animal models?

For in vivo studies of CCDC113:

• Model selection:

  • Xenograft models: Useful for cancer studies as demonstrated with CCDC113-knockdown CRC cells

  • Transgenic models: Consider conditional knockouts for developmental studies

  • Zebrafish: Useful for studying ciliary functions due to transparent embryos

• Experimental design considerations:

  • For tumor studies: Use both subcutaneous xenograft and metastasis models (e.g., tail vein injection)

  • Sample size calculation based on expected effect size

  • Blinded assessment of outcomes

  • Appropriate controls (e.g., shNC vs. shCCDC113)

• Analysis techniques:

  • Tumor volume/weight measurements

  • Histological analysis (H&E staining)

  • IHC for CCDC113 expression and proliferation markers (e.g., Ki67)

  • Metastasis quantification

In published colorectal cancer research, CCDC113 knockdown significantly reduced tumor volume and weight in subcutaneous xenograft models and decreased liver metastasis in tail vein models .

How can researchers investigate CCDC113's role in the ciliary assembly pathway?

To investigate CCDC113's role in ciliary assembly:

• Primary cilium induction protocols:

  • Serum starvation (0.5% FBS for 24-48 hours)

  • Contact inhibition in confluent cultures

  • Specialized media for specific cell types

• Ciliary structure analysis:

  • Immunofluorescence for ciliary markers (acetylated α-tubulin, ARL13B)

  • Transmission electron microscopy for ultrastructural analysis

  • Live-cell imaging with fluorescently tagged ciliary proteins

• Molecular interaction studies:

  • Co-immunoprecipitation to identify CCDC113 binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry analysis of CCDC113 complexes

• Functional readouts:

  • Ciliary length measurements

  • Ciliary signaling pathway activation (Hedgehog, Wnt)

  • Ciliary trafficking assays

Since CCDC113 is a component of centriolar satellites contributing to primary cilium formation, these approaches can help elucidate its specific role in the molecular pathway of ciliogenesis .

What techniques are available for studying the interaction between CCDC113 and other centriolar satellite proteins?

To study CCDC113 interactions with other centriolar satellite proteins:

• Biochemical approaches:

  • Co-immunoprecipitation with anti-CCDC113 antibodies followed by mass spectrometry

  • GST pull-down assays with recombinant CCDC113

  • Yeast two-hybrid screening

  • In vitro binding assays with purified proteins

• Imaging approaches:

  • Dual-color super-resolution microscopy

  • FRET (Förster Resonance Energy Transfer) for direct interactions

  • Proximity ligation assay (PLA) for in situ detection of protein interactions

  • Live-cell imaging with fluorescently tagged proteins

• Functional validation:

  • Mutational analysis of interaction domains

  • Domain mapping through truncation constructs

  • Competition assays with peptides

  • CRISPR-mediated knockout of interaction partners

These techniques can reveal the protein interaction network of CCDC113 at centriolar satellites and help understand how these interactions contribute to cilium formation and function .

How can researchers distinguish between the roles of different CCDC113 isoforms?

To investigate isoform-specific functions of CCDC113:

• Isoform identification:

  • RNA-seq analysis to identify expressed transcripts

  • RT-PCR with isoform-specific primers

  • Western blot analysis with antibodies recognizing different epitopes

• Isoform-specific tools:

  • Design of isoform-specific siRNAs/shRNAs

  • Generation of isoform-specific antibodies

  • Construction of expression vectors for individual isoforms

• Functional analysis:

  • Rescue experiments with individual isoforms in knockdown cells

  • Localization studies of each isoform

  • Protein-protein interaction analysis for each isoform

  • Domain mapping to identify functional differences

• Tissue/cell-specific expression:

  • qRT-PCR with isoform-specific primers across tissues

  • Single-cell RNA-seq to identify cell populations expressing specific isoforms

Since up to two different isoforms have been reported for CCDC113 in humans, understanding their differential expression and function may provide insights into tissue-specific roles of this protein .

What methods are recommended for investigating CCDC113's role in cancer progression?

For investigating CCDC113 in cancer:

• Expression analysis:

  • Mining public datasets (TCGA, GEO) for expression correlations with clinical outcomes

  • Tissue microarray analysis of CCDC113 expression in tumor vs. normal tissues

  • Single-cell RNA-seq to identify expressing cell populations within tumors

• Functional studies:

  • Proliferation assays: CCK-8, colony formation

  • Migration/invasion assays: Wound healing, transwell, 3D invasion models

  • Apoptosis assays: Annexin V/PI staining, caspase activation

  • Cell cycle analysis: Flow cytometry with PI staining

• Mechanistic investigations:

  • RNA-seq after CCDC113 modulation to identify affected pathways

  • ChIP-seq to identify transcription factors regulating CCDC113

  • Phosphoproteomics to identify signaling pathways affected

• In vivo models:

  • Xenograft models with CCDC113-modulated cells

  • Metastasis models (tail vein injection, orthotopic implantation)

  • Patient-derived xenografts (PDX)

Recent research has shown that CCDC113 is highly expressed in colorectal cancer and its knockdown inhibits proliferation and migration in vitro while reducing tumor growth and metastasis in vivo .

How can researchers assess CCDC113's potential as a diagnostic biomarker?

To evaluate CCDC113 as a biomarker:

• Clinical sample analysis:

  • Retrospective analysis of tissue samples with IHC

  • Evaluation in liquid biopsies (circulating tumor cells, exosomes)

  • Correlation with clinicopathological features and outcomes

• Biomarker performance assessment:

  • Sensitivity and specificity calculations

  • ROC curve analysis

  • Comparison with established biomarkers

  • Multivariate analysis to assess independent prognostic value

• Validation approaches:

  • Independent cohort validation

  • Multi-center studies

  • Prospective clinical trials

• Technical considerations:

  • Standardization of detection methods

  • Establishment of positivity thresholds

  • Assessment of pre-analytical variables

CCDC113 has been predicted to be a biomarker for early lung cancer detection and has shown prognostic value in colorectal cancer, suggesting its potential utility as a clinical biomarker .

What experimental designs are appropriate for studying CCDC113's involvement in ciliopathies?

To investigate CCDC113 in ciliopathies:

• Patient-derived materials:

  • Genetic screening of ciliopathy patients for CCDC113 mutations

  • Fibroblast cultures from patients for functional studies

  • Induced pluripotent stem cells (iPSCs) from patients

• Model systems:

  • Zebrafish morpholinos or CRISPR for developmental phenotypes

  • Mouse models with conditional CCDC113 knockout

  • Organoid cultures (kidney, brain, retina) to model tissue-specific ciliary defects

• Functional assays:

  • Ciliary length and frequency measurements

  • Ciliary beat frequency analysis

  • Ciliary protein trafficking assays

  • Hedgehog and Wnt signaling reporter assays

• Structural analysis:

  • Super-resolution microscopy of cilia

  • Transmission electron microscopy for ultrastructural analysis

  • Cryogenic electron microscopy for molecular details

Since CCDC113 contributes to primary cilium formation, disruptions in its function may contribute to ciliopathies, which are genetic disorders resulting from defects in ciliary structure or function .

How can researchers address non-specific binding when using CCDC113 antibodies?

To minimize non-specific binding:

• Antibody validation:

  • Test on CCDC113-knockout/knockdown samples as negative controls

  • Compare multiple antibodies targeting different epitopes

  • Pre-adsorption tests with blocking peptides

• Optimization strategies:

  • Titrate antibody concentration (typically 1:500-1:2000 for Western blot)

  • Optimize blocking conditions (5% BSA often reduces background compared to milk)

  • Increase washing duration and frequency

  • Consider alternative buffers (PBST vs. TBST)

• Sample-specific considerations:

  • Pre-clear lysates to remove proteins that bind non-specifically

  • Use tissue-specific blocking agents

  • Consider antigen retrieval methods for fixed tissues

• Signal enhancement without increasing background:

  • Use high-sensitivity detection systems

  • Consider signal amplification methods like tyramide signal amplification

These approaches can help ensure specific detection of CCDC113 and minimize false positive results in experimental applications .

What are the recommended protocols for quantitative analysis of CCDC113 expression levels?

For quantitative CCDC113 expression analysis:

• RNA-level quantification:

  • qRT-PCR with validated primers (efficiency >95%)

  • Standard curve method or ΔΔCt with validated reference genes

  • Digital droplet PCR for absolute quantification

  • RNA-seq with appropriate normalization methods

• Protein-level quantification:

  • Western blot with housekeeping protein controls

  • ELISA with recombinant protein standards

  • Mass spectrometry with labeled standards

  • Quantitative immunofluorescence with calibration standards

• Analysis considerations:

  • Include multiple biological and technical replicates

  • Use appropriate statistical tests based on data distribution

  • Consider power analysis to determine sample size

  • Account for batch effects in large studies

• Data representation:

  • Normalized expression values relative to controls

  • Fold-change with appropriate error bars

  • Box plots or violin plots for distribution visualization

These methodologies have been successfully applied in studies examining CCDC113 expression in colorectal cancer and other contexts .

How should researchers interpret contradictory results from different CCDC113 antibodies?

When facing contradictory results:

• Systematic validation approach:

  • Compare antibody specifications (epitope location, host species, clonality)

  • Validate each antibody using positive and negative controls

  • Test multiple lots of the same antibody

  • Conduct side-by-side comparisons under identical conditions

• Technical considerations:

  • Evaluate whether discrepancies are application-specific

  • Assess potential post-translational modifications affecting epitope recognition

  • Consider isoform-specific detection as a source of variation

  • Evaluate sample preparation differences

• Alternative validation methods:

  • Genetic approaches (CRISPR knockout, siRNA) to verify specificity

  • Mass spectrometry to confirm protein identity

  • Epitope mapping to understand binding differences

  • Orthogonal methods that don't rely on antibodies

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Report results with appropriate caveats

  • Consider consortium approaches for antibody validation

Contradictory results may reveal important biological insights about isoforms, post-translational modifications, or protein interactions that affect epitope accessibility .

What methodologies are appropriate for investigating CCDC113's potential roles beyond ciliary assembly?

To explore novel CCDC113 functions:

• Unbiased screening approaches:

  • Interactome analysis via BioID or APEX proximity labeling

  • Phosphoproteomic analysis after CCDC113 modulation

  • CRISPR screens in CCDC113-modulated backgrounds

  • Yeast two-hybrid or mammalian two-hybrid screens

• Subcellular localization studies:

  • Fractionation followed by Western blotting

  • High-resolution imaging with co-localization analysis

  • Live-cell imaging during different cellular processes

  • Electron microscopy with immunogold labeling

• Functional readouts:

  • Cell cycle analysis with synchronized cells

  • DNA damage response assays

  • Stress response measurements

  • Metabolic pathway analysis

• Context-dependent studies:

  • Analysis across different cell types

  • Response to various stimuli or stressors

  • Developmental stage-specific analysis

CCDC113's reported associations with diverse conditions including post-stroke cognitive impairment, asthma, and cancer suggest functions beyond its established role in ciliary assembly .

How can researchers investigate the relationship between CCDC113 and post-stroke cognitive impairment?

To study CCDC113 in post-stroke cognitive impairment:

• Clinical approaches:

  • Genetic association studies in stroke patient cohorts

  • Protein/gene expression analysis in patient samples

  • Correlation of CCDC113 levels with cognitive assessment scores

  • Longitudinal studies tracking expression changes and outcomes

• In vitro models:

  • Oxygen-glucose deprivation (OGD) in neuronal cultures

  • CCDC113 modulation in neuronal or glial cells

  • Blood-brain barrier models with endothelial cells

  • Co-culture systems mimicking neurovascular units

• In vivo models:

  • Middle cerebral artery occlusion (MCAO) mouse models

  • Cognitive testing after stroke in CCDC113-modulated animals

  • Neuroimaging combined with molecular analysis

  • Tissue-specific conditional knockout models

• Mechanistic investigations:

  • Inflammatory pathway analysis

  • Synaptic plasticity assessments

  • Neurovascular coupling measurements

  • Blood-brain barrier integrity evaluation

Bioinformatics analysis has shown that CCDC113 is associated with post-stroke cognitive impairment, suggesting a novel function beyond its known roles in ciliary biology .

What experimental approaches could elucidate CCDC113's potential role in asthma pathogenesis?

To investigate CCDC113 in asthma:

• Clinical studies:

  • Expression analysis in bronchial biopsies from asthma patients

  • Genetic association studies in asthma cohorts

  • Protein levels in bronchoalveolar lavage fluid

  • Single-cell RNA-seq of airway samples

• In vitro models:

  • Air-liquid interface cultures of bronchial epithelial cells

  • CCDC113 modulation in airway epithelial cells

  • Ciliary beat frequency analysis

  • Mucus production and clearance assays

• In vivo approaches:

  • Asthma models in CCDC113-modulated mice

  • Airway hyperresponsiveness measurements

  • Inflammatory cell influx quantification

  • Airway remodeling assessment

• Mechanistic studies:

  • Type 2 inflammation pathway analysis

  • Epithelial barrier function tests

  • Ciliary function assessments

  • Mucociliary clearance measurements

CCDC113 has been associated with asthma in bioinformatics analyses, and given its role in ciliary function, it may contribute to airway epithelial dysfunction in asthma pathogenesis .