ccdc113 Antibody

What is CCDC113 and what cellular functions is it associated with?

CCDC113 is a cytoplasmic protein with a canonical length of 377 amino acid residues and a molecular weight of 44.2 kDa in humans. It functions as a component of centriolar satellites that contribute to primary cilium formation. Up to two different isoforms have been reported for this protein. CCDC113 has been implicated in several biological processes including ciliary assembly and ciliary activity. Recent research has associated CCDC113 with post-stroke cognitive impairment (PSCI), asthma, and early lung cancer diagnosis. Most significantly, CCDC113 has been identified as promoting colorectal cancer tumorigenesis and metastasis, potentially through mechanisms involving transmembrane transport and Wnt signaling pathways .

How does CCDC113 expression vary across different tissue types and disease states?

CCDC113 expression shows significant variation between normal and pathological states:

• Normal tissues: CCDC113 is expressed in cells with ciliary structures, consistent with its role in ciliary assembly.

• Colorectal cancer (CRC): CCDC113 shows significantly higher expression in CRC tissues compared to normal colonic epithelial cells.

• Cell line expression patterns: Among colorectal cancer cell lines, HCT116 and RKO cells exhibit relatively higher CCDC113 expression levels compared to other lines like SW480, HT29, SW620, and LoVo.

• Subcellular distribution: Immunofluorescence studies demonstrate that CCDC113 predominantly localizes to the cytoplasm in colorectal cancer cells.

• Prognostic significance: High expression of CCDC113 correlates with poor prognosis in colorectal cancer patients, indicating its potential value as a prognostic biomarker .

Understanding these expression patterns is crucial for experimental design and result interpretation when using CCDC113 antibodies in research contexts.

What are the most validated applications for CCDC113 antibodies in research?

CCDC113 antibodies have been validated for several key research applications:

• Western Blot (WB): Widely used for detecting CCDC113 protein expression levels and confirming molecular weight (44.2 kDa).

• Enzyme-Linked Immunosorbent Assay (ELISA): Commonly employed for quantitative detection of CCDC113 in various sample types.

• Immunohistochemistry (IHC): For examining tissue distribution and cellular localization of CCDC113 in fixed samples.

• Immunofluorescence (IF): Particularly useful for visualizing CCDC113's predominantly cytoplasmic localization at the subcellular level.

Several CCDC113 antibodies have been cited in research publications, providing evidence of their reliability in these applications. When selecting an antibody, researchers should review validation data, including figures demonstrating application-specific performance .

What criteria should guide the selection of a CCDC113 antibody for specific research applications?

When selecting a CCDC113 antibody, researchers should consider multiple factors:

• Experimental application compatibility: Different antibodies perform optimally in specific applications (WB, IHC, ELISA, IF), so select one validated for your intended use.

• Species reactivity: Ensure the antibody recognizes CCDC113 in your species of interest. CCDC113 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species.

• Antibody format: Choose between unconjugated antibodies or those conjugated to reporter molecules based on your detection method.

• Validation evidence: Review literature citations and validation figures provided by suppliers (67 CCDC113 antibodies across 14 suppliers are commercially available).

• Epitope specificity: Consider which protein domain is most relevant to your research question, especially if studying specific domains or distinguishing between isoforms.

• Polyclonal vs. monoclonal: Evaluate the trade-off between the broader epitope recognition of polyclonals versus the higher specificity of monoclonals.

For critical applications, testing multiple antibodies targeting different CCDC113 epitopes is recommended to ensure reliable results .

What synonyms and alternative names for CCDC113 should researchers be aware of when searching literature?

When conducting literature searches related to CCDC113, researchers should be aware of several synonyms and alternative designations:

• HSPC065: An alternative gene symbol sometimes used in older literature

• Coiled-coil domain-containing protein 113: The full protein name

• CCD113: An abbreviated form occasionally used

• C16orf51: A historical designation based on chromosomal location (chromosome 16 open reading frame 51)

Additionally, researchers should be aware that CCDC113 belongs to the broader coiled-coil domain containing (CCDC) protein family, which includes numerous members with diverse functions. In database searches, using multiple identifiers will ensure comprehensive results, particularly when exploring older literature or specialized databases .

What are the optimal fixation and permeabilization protocols for CCDC113 immunodetection?

Successful immunodetection of CCDC113 depends on appropriate sample preparation protocols:

For immunofluorescence in cell cultures:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature preserves CCDC113 epitopes while maintaining cellular architecture

• Permeabilization: 0.1-0.2% Triton X-100 for 5-10 minutes allows antibody access to cytoplasmic CCDC113

• Blocking: 5% normal serum (from the species in which the secondary antibody was raised) for 1 hour at room temperature

For immunohistochemistry in tissue sections:

• Fixation: 10% neutral buffered formalin followed by paraffin embedding

• Sectioning: 4-6 μm sections mounted on positively charged slides

• Antigen retrieval: Citrate buffer (pH 6.0) heat-induced epitope retrieval enhances CCDC113 detection

• Permeabilization: 0.3% Triton X-100 in PBS for 15-20 minutes

• Blocking: 5-10% normal serum plus 1% BSA in PBS

These conditions should be optimized for each specific CCDC113 antibody and experimental system, as slight modifications may be required for optimal results .

How should I optimize western blot conditions for detecting CCDC113?

For optimal western blot detection of CCDC113, researchers should consider the following protocol:

Sample preparation:

• Lysis buffer: RIPA buffer supplemented with protease inhibitors effectively extracts CCDC113

• Loading amount: 20-40 μg of total protein per lane is typically sufficient

• Denaturation: 95°C for 5 minutes in reducing sample buffer

Gel electrophoresis and transfer:

• Gel percentage: 10-12% SDS-PAGE gels provide good resolution for the 44.2 kDa CCDC113 protein

• Running conditions: 100-120V constant voltage

• Transfer: Wet transfer at 100V for 60-90 minutes to PVDF membrane

Antibody incubation:

• Primary antibody: Typical dilutions range from 1:500 to 1:2000 depending on the specific antibody

• Incubation: Overnight at 4°C generally yields the best signal-to-noise ratio

• Secondary antibody: HRP-conjugated antibodies at 1:5000-1:10000 dilution for 1 hour at room temperature

Detection and expected results:

• Enhanced chemiluminescence (ECL) detection systems provide good sensitivity

• Expected band: The canonical CCDC113 protein should appear at approximately 44.2 kDa

• Positive controls: Include lysates from cells known to express CCDC113, such as HCT116 or RKO colorectal cancer cells

If detecting multiple bands, consider the possibility of isoforms, post-translational modifications, or proteolytic processing of CCDC113 .

What controls are essential when using CCDC113 antibodies for immunohistochemistry?

Rigorous controls are critical for reliable CCDC113 immunohistochemistry experiments:

Positive tissue controls:

• Colorectal cancer tissue samples known to express CCDC113

• Cell lines with confirmed CCDC113 expression (e.g., HCT116, RKO cells) prepared as cell blocks

• Tissue microarrays containing multiple samples for standardized comparison

Negative controls:

• No primary antibody control: To assess non-specific binding of the secondary antibody

• Isotype control: Primary antibody replaced with non-specific IgG of the same isotype and concentration

• Absorption control: Primary antibody pre-incubated with excess CCDC113 peptide/protein

• CCDC113 knockdown/knockout samples: Tissue or cells with verified reduction of CCDC113

Technical standardization:

• Internal positive controls within the same tissue section to normalize staining intensity

• Consistent staining protocols with documented lot numbers and dilutions

• Standardized image acquisition settings for comparative studies

• Digital image analysis with validated algorithms when performing quantitative assessment

Additionally, researchers should validate staining patterns by comparing results with published literature and confirming the predominantly cytoplasmic localization pattern of CCDC113 .

How can I validate the specificity of my CCDC113 antibody?

Validating CCDC113 antibody specificity requires multiple complementary approaches:

Genetic validation approaches:

• Compare staining in cells with and without CCDC113 knockdown (using shRNA as demonstrated in colorectal cancer studies)

• Test the antibody in CCDC113 knockout models if available

• Examine increased signal in cells overexpressing CCDC113 (as shown in CRC overexpression models)

Molecular validation methods:

• Confirm a single band at the expected molecular weight (44.2 kDa) by western blot

• Perform peptide competition assays by pre-incubating the antibody with immunizing peptide

• Consider immunoprecipitation followed by mass spectrometry to verify that the recognized protein is indeed CCDC113

Cross-platform validation:

• Compare detection patterns across different techniques (WB, IHC, IF)

• Correlate protein detection with mRNA expression data (RT-PCR or RNA-seq)

• Test multiple antibodies targeting different CCDC113 epitopes

Functional validation:

• Demonstrate that antibody-detected expression changes correspond with functional changes

• Show that antibody detection correlates with expected biological phenomena (e.g., ciliary assembly defects)

What concentrations of CCDC113 antibodies are typically effective for different applications?

Optimal CCDC113 antibody concentrations vary by application and specific antibody characteristics:

Western Blot:

• Typical dilutions: 1:500 to 1:2000 of commercial antibodies

• Concentrated antibodies: 0.2-1 μg/ml final concentration

• Incubation: Overnight at 4°C in 5% BSA or non-fat milk in TBST

Immunohistochemistry:

• FFPE sections: 1:100 to 1:500 dilution

• Frozen sections: 1:200 to 1:1000 dilution

• Typical concentration: 1-5 μg/ml

• Incubation: 1-2 hours at room temperature or overnight at 4°C

Immunofluorescence:

• Cell cultures: 1:200 to 1:500 dilution

• Typical concentration: 2-10 μg/ml

• Incubation: 1-2 hours at room temperature or overnight at 4°C

ELISA:

• Capture antibody: 1-10 μg/ml

• Detection antibody: 0.1-1 μg/ml (if directly conjugated) or 1:1000 to 1:5000 dilution

These recommendations serve as starting points; optimal concentrations should be determined empirically for each experimental system and specific antibody through titration experiments .

Why might I observe multiple bands when detecting CCDC113 by western blot?

Multiple bands on CCDC113 western blots can occur for several biological and technical reasons:

Biological factors:

• Isoforms: CCDC113 has up to two reported isoforms that may appear as distinct bands

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can alter migration patterns

• Proteolytic processing: Partial degradation or specific cleavage of CCDC113 during sample preparation

• Protein complexes: Incomplete denaturation may result in higher molecular weight bands

Technical issues:

• Cross-reactivity: The antibody may recognize proteins with similar epitopes

• Non-specific binding: Insufficient blocking or high antibody concentration can cause background bands

• Sample degradation: Improper sample handling or insufficient protease inhibitors

• Incomplete transfer: Uneven protein transfer to membrane can create artifacts

Troubleshooting approaches:

• Verify with multiple antibodies targeting different CCDC113 epitopes

• Include positive controls (CCDC113 overexpression) and negative controls (CCDC113 knockdown)

• Optimize sample preparation with fresh protease inhibitors and complete denaturing conditions

• Perform peptide competition assays to identify which bands represent specific binding

Understanding the source of multiple bands is crucial for accurate interpretation of CCDC113 expression data in experimental systems .

How can I address background issues in CCDC113 immunostaining experiments?

Background issues in CCDC113 immunostaining can be addressed through systematic optimization:

Causes of background:

• Non-specific binding of primary or secondary antibodies

• Inadequate blocking

• Autofluorescence (in fluorescent detection)

• Endogenous peroxidase activity (in HRP-based detection)

• Cross-reactivity with similar epitopes

• Excessive antibody concentration

Solutions for immunohistochemistry:

• Optimize blocking: Use 5-10% normal serum from the secondary antibody species for 1-2 hours

• Quench endogenous peroxidase: Treat sections with 0.3-3% H₂O₂ before antibody incubation

• Reduce antibody concentration: Titrate to find minimal concentration giving specific signal

• Add blocking proteins: 0.1-0.5% BSA or 0.1-0.3% Triton X-100 in antibody diluent

• Increase washing frequency and duration with PBS-T or TBS-T

• Use avidin/biotin blocking kit if using biotin-based detection systems

Solutions for immunofluorescence:

• Reduce autofluorescence: Treat with 0.1-1% sodium borohydride or commercial quenchers

• Use Sudan Black B (0.1-0.3%): Particularly effective for reducing lipofuscin autofluorescence

• Optimize fixation: Excessive fixation can increase background

• Use fluorophores with spectra distinct from tissue autofluorescence

Always include appropriate controls to distinguish specific staining from background and verify the predominantly cytoplasmic localization pattern expected for CCDC113 .

What strategies can resolve inconsistent CCDC113 antibody performance between experiments?

Inconsistent CCDC113 antibody performance can be addressed through standardization and systematic troubleshooting:

Standardization strategies:

• Create single-use antibody aliquots to avoid freeze-thaw cycles

• Document detailed protocols with exact timings, temperatures, and reagent lots

• Standardize sample processing: fixation times, buffer compositions, and processing steps

• Include identical positive and negative control samples in every experiment

• When possible, prepare reagent mixtures for all samples simultaneously

• Maintain consistent temperature, humidity, and incubation conditions

Technical considerations:

• Store antibodies according to manufacturer recommendations (typically -20°C or -80°C)

• Use freshly prepared buffers and working solutions

• Process all experimental samples simultaneously when possible

• Regularly calibrate imaging systems and laboratory equipment

• Document antibody lot numbers and test new lots against previous ones

• Control for cell confluency, passage number, or tissue preservation method

Validation approaches:

• Cross-validate with multiple detection methods

• Verify with independent antibodies targeting different CCDC113 epitopes

• Correlate with mRNA expression data from the same samples

• Include calibration samples with known CCDC113 expression levels

How should I interpret contradictory CCDC113 expression data between different detection methods?

Interpreting contradictory CCDC113 expression data requires careful consideration of each method's limitations:

Understanding methodological differences:

• Western blot: Measures denatured protein, good for quantifying total expression but loses spatial information

• IHC/IF: Maintains spatial context but may have limited quantitative accuracy

• ELISA: Highly quantitative but requires protein extraction and may detect specific epitopes only

• qRT-PCR/RNA-seq: Measures mRNA, not protein; post-transcriptional regulation may cause discrepancies

Resolution strategies:

• Evaluate technical quality:

  • Assess controls for each method

  • Review antibody validation for each technique

  • Consider signal-to-noise ratio and detection limits

• Consider biological factors:

  • Different isoforms may be detected preferentially by different methods

  • Post-translational modifications may affect epitope accessibility

  • Cytoplasmic localization of CCDC113 may influence extraction efficiency

  • Protein stability and turnover rates may differ from mRNA

• Reconciliation approaches:

  • Use multiple antibodies targeting different CCDC113 epitopes

  • Employ complementary techniques (e.g., immunoprecipitation followed by mass spectrometry)

  • Conduct genetic manipulation studies (overexpression, knockdown) to validate findings

  • Consider single-cell analyses to address population heterogeneity

When reporting contradictory findings, clearly state the methods used and discuss potential reasons for discrepancies to advance understanding of CCDC113 biology .

What are common pitfalls when quantifying CCDC113 expression in tumor samples?

Quantifying CCDC113 expression in tumor samples involves several potential pitfalls that researchers should address:

Tumor heterogeneity challenges:

• Intratumoral heterogeneity: CCDC113 expression may vary within different regions of the same tumor

• Stromal contamination: Non-tumor cells within the sample may dilute or confound expression measurements

• Necrotic areas: Dead or dying cells can produce artifacts or reduced signal

• Variable cellularity: Differences in cell density can affect quantification

Technical challenges:

• Fixation artifacts: Overfixation or delayed fixation can affect epitope availability

• Batch effects: Processing multiple samples across different days can introduce variation

• Antibody specificity: Cross-reactivity with related proteins

• Threshold determination: Subjective cutoffs for "high" versus "low" expression

• Quantification method differences: H-score, Allred score, or digital image analysis may yield different results

Recommendations:

• Use tissue microarrays for standardized processing when comparing multiple samples

• Implement digital pathology with validated algorithms for objective quantification

• Include multiple tumor regions to address heterogeneity

• Validate findings with orthogonal methods (e.g., WB, qRT-PCR)

• Correlate with clinical parameters and survival data as demonstrated in colorectal cancer studies

• Consider the prognostic significance of CCDC113 expression in the context of other biomarkers

Understanding these pitfalls is essential for generating reliable data on CCDC113 expression in tumor samples, particularly when evaluating its potential as a prognostic biomarker in colorectal cancer .

How can CCDC113 antibodies be employed to investigate its role in ciliary assembly?

CCDC113 antibodies can be strategically employed to elucidate its role in ciliary assembly through multiple sophisticated approaches:

Colocalization studies:

• Double immunofluorescence staining with CCDC113 antibodies and established ciliary markers (acetylated tubulin, IFT88, Arl13b)

• Super-resolution microscopy to precisely map CCDC113 localization within centriolar satellites

• Live-cell imaging with fluorescently tagged CCDC113 antibody fragments to track dynamics during ciliogenesis

Protein interaction analyses:

• Immunoprecipitation with CCDC113 antibodies followed by mass spectrometry to identify ciliary assembly interactors

• Proximity ligation assays to confirm direct interactions with other centriolar satellite components

• Immunoblotting for CCDC113 in fractionated cellular components during different stages of cilium formation

Perturbation experiments:

• Combine CCDC113 knockdown/knockout with immunostaining for ciliary markers to assess structural defects

• Perform rescue experiments with mutant CCDC113 constructs followed by antibody-based detection of ciliary phenotypes

• Use function-blocking antibodies to disrupt specific CCDC113 interactions during cilium assembly

Developmental and disease models:

• Track CCDC113 expression and localization during developmental stages of cilium formation

• Compare CCDC113 distribution in normal versus disease models with ciliary defects

• Correlate CCDC113 expression patterns with ciliary morphology in patient samples

These approaches can provide mechanistic insights into how CCDC113 contributes to centriolar satellite function and primary cilium formation, which may have implications for related pathologies .

What techniques can elucidate CCDC113's interactions with other proteins in the Wnt signaling pathway?

To investigate CCDC113's interactions with Wnt signaling pathway components, researchers can employ several sophisticated techniques:

Co-immunoprecipitation approaches:

• Reciprocal co-IP using antibodies against CCDC113 and key Wnt pathway components

• Tandem affinity purification with tagged CCDC113 followed by mass spectrometry

• Proximity-dependent biotin identification (BioID) by fusing CCDC113 to a biotin ligase

Protein-protein interaction visualization:

• Förster resonance energy transfer (FRET) between fluorescently labeled CCDC113 and Wnt pathway components

• Proximity ligation assay (PLA) using CCDC113 antibodies with antibodies against Wnt pathway proteins

• Immunofluorescence colocalization with super-resolution microscopy for precise spatial relationships

Functional interaction studies:

• Luciferase reporter assays measuring TCF/LEF transcriptional activity after CCDC113 manipulation

• CRISPR/Cas9 knockouts of CCDC113 and Wnt components to assess pathway dependencies

• Small molecule perturbation using Wnt pathway activators/inhibitors while monitoring CCDC113 interactions

Bioinformatic approaches:

• Integration of proteomic data with known Wnt pathway interactomes

• Analysis of co-expression patterns across tissues and disease states

• Pathway enrichment analysis as performed in colorectal cancer studies

These techniques can help establish whether CCDC113 directly interacts with Wnt signaling components or influences the pathway through indirect mechanisms, potentially explaining its role in colorectal cancer progression .

How can I design experiments to explore CCDC113's potential as a biomarker in colorectal cancer?

Designing experiments to evaluate CCDC113 as a colorectal cancer biomarker requires a comprehensive, multi-stage approach:

Clinical sample analysis:

• Retrospective cohort study: Analyze CCDC113 expression in archived CRC samples with known clinical outcomes

• Tissue microarray evaluation: Screen large patient cohorts for CCDC113 expression patterns

• Liquid biopsy development: Assess if CCDC113 protein or antibodies against it are detectable in patient serum

• Paired sample comparison: Analyze matched normal-tumor pairs to establish tumor specificity

Analytical validation:

• Antibody validation: Test multiple CCDC113 antibodies for specificity and reproducibility

• Multi-platform confirmation: Compare IHC, western blot, and ELISA for consistent detection

• Analytical sensitivity determination: Establish limits of detection for various sample types

• Inter-laboratory validation: Confirm reproducibility across different research settings

Clinical correlation studies:

• Correlation with established CRC biomarkers (CEA, CA19-9)

• Association with clinicopathological parameters (tumor stage, grade, invasion depth)

• Survival analysis: Kaplan-Meier analysis comparing high vs. low CCDC113 expression groups

• Treatment response prediction: Correlation of CCDC113 levels with therapy outcomes

Functional validation:

• Knockout/knockdown studies: Assess phenotypic changes in CRC models as demonstrated in published research

• Overexpression experiments: Determine if CCDC113 overexpression confers malignant properties

• Mechanistic studies: Investigate pathways through which CCDC113 promotes CRC progression

• Animal models: Validate findings in xenograft models as shown in recent research

Existing research has already demonstrated that high CCDC113 expression correlates with poor prognosis in CRC patients and that it influences both tumorigenesis and metastasis, providing a strong foundation for its further development as a biomarker .

What methodological approaches can investigate the role of CCDC113 in cancer metastasis?

CCDC113 antibodies can be strategically employed to investigate its role in metastasis through multiple experimental approaches:

In vitro metastatic models:

• Immunofluorescence tracking of CCDC113 localization during epithelial-mesenchymal transition (EMT)

• Western blot comparison of CCDC113 levels between metastatic and non-metastatic cell lines

• Correlation of CCDC113 expression with invasive capacity using antibody staining in functional assays

• Transwell migration and wound-healing assays with CCDC113 knockdown or overexpression

In vivo metastasis models:

• IHC of primary tumors and metastatic lesions: Compare CCDC113 expression patterns

• Xenograft models: Immunostaining of CCDC113 in metastatic nodules from different organs

• Tail vein metastasis model: As demonstrated in published research, CCDC113 knockdown significantly reduced liver metastasis while overexpression increased metastatic capacity

Human patient samples:

• Paired primary-metastatic tumor analysis: Compare CCDC113 expression and localization

• Tissue microarray screening: Correlate CCDC113 levels with metastatic status across patient cohorts

• Prognostic evaluation: Correlate CCDC113 expression with metastasis-free survival

Mechanistic investigations:

• Co-immunoprecipitation to identify CCDC113 interactions with known metastasis regulators

• Protein-protein interaction network analysis as performed in colorectal cancer studies

• GO enrichment analysis of CCDC113 interacting proteins, which has revealed significant enrichment in various transmembrane transporter activities

These approaches can build upon the existing evidence that CCDC113 plays a significant role in colorectal cancer metastasis, potentially leading to the development of new therapeutic strategies targeting this process .

How can CCDC113 antibodies be used to study its involvement in primary cilium-related pathologies?

CCDC113's role in ciliary assembly makes it relevant to various ciliopathies, and antibodies can be employed to study this connection:

Ciliopathy model systems:

• Patient-derived cells: Compare CCDC113 expression and localization in cells from ciliopathy patients versus healthy controls

• Genetic ciliopathy models: Examine CCDC113 distribution in animal models of ciliopathies

• Induced pluripotent stem cells (iPSCs): Differentiate into ciliated cells to study CCDC113 during ciliogenesis

Structural analysis:

• Super-resolution microscopy: Map CCDC113 within the ciliary structure using labeled antibodies

• Transmission electron microscopy with immunogold labeling: Precisely localize CCDC113 at the ultrastructural level

• Live imaging: Track CCDC113 dynamics during cilium formation and function

Functional studies:

• Ciliary signaling analysis: Examine how CCDC113 manipulation affects cilium-dependent signaling pathways

• Flow sensing: Measure ciliary responses to flow with and without CCDC113 perturbation

• Ciliary trafficking: Track intraflagellar transport after CCDC113 knockdown or overexpression

Clinical correlations:

• Tissue analysis: Compare CCDC113 expression in tissues affected by ciliopathies

• Genotype-phenotype correlation: Relate CCDC113 mutations or expression changes to clinical manifestations

• Therapeutic response: Monitor CCDC113 as a biomarker during treatment of ciliopathy-related conditions

These approaches could provide insights into whether CCDC113 dysfunction contributes to ciliopathies and whether targeting CCDC113 might have therapeutic potential in these disorders .