CCDC6 Antibody

What is the optimal antibody selection for detecting CCDC6 in different experimental contexts?

When selecting a CCDC6 antibody, researchers should consider several factors including the host species, clonality, and validated applications. For western blot applications, rabbit polyclonal antibodies such as CAB16075 have demonstrated high specificity for human CCDC6 with recommended dilutions of 1:500-1:2000 . For immunohistochemical analysis, antibodies like anti-CCDC6 (HPA 019051) from Sigma-Aldrich have shown reliable results in paraffin-embedded tissues . The selection should be guided by:

• Target species compatibility (human, mouse, rat)

• Experimental technique (WB, IHC-P, IF/ICC, ELISA)

• Cellular localization needs (cytoplasm, cytoskeleton)

• Molecular weight detection requirements (calculated MW: 53kDa; observed MW: 56kDa/70kDa)

For consistent results across multiple studies, document the antibody catalog number, lot, and working dilution in your laboratory protocols.

How can I troubleshoot weak or absent CCDC6 signal in western blot analysis?

When encountering weak or absent CCDC6 signals in western blots, consider the following methodological approaches:

• Sample preparation optimization: CCDC6 stability is dependent on USP7 de-ubiquitinase activity. Treating cells with proteasome inhibitors prior to lysis may prevent degradation and enhance detection .

• Antibody concentration adjustment: If using polyclonal antibodies like CAB16075, test a higher concentration range (1:500 instead of 1:2000) while monitoring background signal .

• Protein loading assessment: CCDC6 expression varies significantly between cell types. GC-1 spermatogonia and NTERA-2 embryonal carcinoma cells show appreciable CCDC6 expression, while GC-2 spermatocytes and TM4 Sertoli cells exhibit nearly undetectable levels .

• Detection system sensitivity: Consider using enhanced chemiluminescence substrates or fluorescence-based systems for low-abundance CCDC6 detection.

• Post-translational modifications: Be aware that CCDC6 may display varying molecular weights (56kDa/70kDa observed vs. 53kDa calculated) due to phosphorylation and other modifications .

What are the recommended positive control samples for validating CCDC6 antibodies?

Based on published research, the following samples serve as reliable positive controls for CCDC6 antibody validation:

When validating a new CCDC6 antibody, comparing your results with published expression patterns in these established models provides confidence in antibody specificity and sensitivity.

How can CCDC6 antibodies be used to investigate DNA damage response pathways?

CCDC6 antibodies serve as valuable tools for investigating DNA damage response (DDR) pathways through several methodological approaches:

• Homologous recombination (HR) efficiency assessment: Following CCDC6 protein detection via immunoblotting, researchers can correlate CCDC6 levels with HR efficiency using DR-GFP reporter assays. CCDC6 deficiency has been demonstrated to impair HR mechanisms, which can be quantified by flow cytometry measuring the percentage of GFP-positive cells following induced DNA double-strand breaks .

• Co-immunoprecipitation studies: CCDC6 antibodies can identify interactions with DDR proteins following DNA damage. Precipitation of CCDC6 followed by immunoblotting for DNA repair factors enables mapping of protein complexes formed during the repair process.

• Chromatin immunoprecipitation (ChIP): CCDC6 antibodies can be used in ChIP assays to determine CCDC6 recruitment to sites of DNA damage, providing insights into its direct role in DNA repair mechanisms.

• Immunofluorescence microscopy: Detecting CCDC6 co-localization with γH2AX foci or RAD51 can reveal temporal dynamics of CCDC6 involvement in DNA damage signaling and repair. Research has shown that low CCDC6 protein levels correlate with reduced RAD51 foci formation following damage .

These approaches collectively enable comprehensive mapping of CCDC6's role in maintaining genomic integrity through DNA damage response regulation.

What methodologies are recommended for investigating CCDC6 post-translational modifications using antibodies?

Investigation of CCDC6 post-translational modifications requires specialized approaches:

• Phospho-specific antibody application: CCDC6 function is regulated through phosphorylation. When studying CCDC6 phosphorylation status, researchers should:

  • Treat cells with phosphatase inhibitors during lysis

  • Run parallel immunoblots with phospho-specific and total CCDC6 antibodies

  • Validate phosphorylation sites through lambda phosphatase treatment controls

• Ubiquitination analysis: CCDC6 stability is regulated through ubiquitination-dependent degradation. To study this:

  • Co-transfect cells with tagged-ubiquitin and CCDC6 constructs

  • Immunoprecipitate with CCDC6 antibodies like anti-CCDC6 (ab56353)

  • Detect ubiquitinated species through immunoblotting

  • Compare wild-type CCDC6 with mutant variants (e.g., CCDC6T434A)

• Half-life determination: To measure CCDC6 protein stability:

  • Treat cells with cycloheximide to inhibit protein synthesis

  • Collect samples at defined time points

  • Perform immunoblotting with CCDC6 antibodies

  • Quantify protein levels relative to loading controls

  • Calculate half-life through regression analysis

This approach has demonstrated that USP7 inhibition with P5091 reduces CCDC6 half-life, indicating destabilization through increased proteasomal degradation .

How can CCDC6 antibodies be utilized to stratify cancer patients for PARP inhibitor therapy?

CCDC6 antibodies offer potential for patient stratification for PARP inhibitor therapy through:

• Immunohistochemical scoring systems:

  • Analyze CCDC6 expression in tissue microarrays using validated antibodies

  • Quantify staining intensity on a 0-3 scale

  • Determine percentage of positive tumor cells

  • Calculate H-scores (intensity × percentage)

  • Correlate with clinical outcomes

• Predictive biomarker validation:
  Research has demonstrated that CCDC6 deficiency sensitizes cancer cells to PARP inhibitors (e.g., olaparib) while conferring resistance to traditional therapeutics like cisplatin . This differential response pattern suggests CCDC6 expression levels could predict therapy response.

• Combined analysis with other DDR markers:
  For improved predictive value, CCDC6 immunostaining should be analyzed alongside other homologous recombination deficiency markers. CCDC6 protein expression has been found to correlate with RAD51 foci formation capacity, which directly influences PARP inhibitor sensitivity .

How should experiments be designed to investigate the relationship between CCDC6 expression and reactive oxygen species (ROS) tolerance?

When investigating CCDC6's role in ROS tolerance, researchers should implement the following experimental design:

• Establish CCDC6 expression models:

  • Generate CCDC6-deficient cells through shRNA (e.g., using pLKO.1 puro vectors targeting CCDC6/Ccdc6)

  • Create CCDC6-overexpressing models using expression vectors (e.g., pcDNA4ToA-mycCCDC6wt)

  • Include appropriate controls (empty vector, non-targeting shRNA)

  • Validate CCDC6 modulation through western blotting

• ROS challenge assays:

  • Expose cells to graduated hydrogen peroxide concentrations

  • Measure intracellular ROS using fluorescent probes

  • Quantify cell viability through MTT/XTT assays

  • Assess apoptosis via Annexin V/PI staining

  • Evaluate ferroptosis markers (e.g., lipid peroxidation)

• Mechanistic investigation:

  • Analyze xCT/SLC7A11 cystine antiporter expression via western blot

  • Measure glutathione levels in CCDC6-deficient versus proficient cells

  • Evaluate ferroptosis sensitivity through RSL3 or erastin treatment

  • Rescue experiments with antioxidants or cysteine supplementation

Research has demonstrated that CCDC6 deficiency confers tolerance to oxidative damage through enhanced expression and activity of the xCT/SLC7A11 cystine antiporter, leading to evasion of regulated cell death pathways including apoptosis and ferroptosis . This experimental approach allows for comprehensive characterization of CCDC6's role in redox homeostasis.

What controls are essential when using CCDC6 antibodies for studying DNA repair defects?

When investigating DNA repair defects using CCDC6 antibodies, implement these essential controls:

• Antibody validation controls:

  • Positive controls: Cell lines with known CCDC6 expression (e.g., MCF7, HeLa)

  • Negative controls: CCDC6-depleted cells via siRNA/shRNA

  • Peptide competition assays to confirm antibody specificity

  • Secondary antibody-only controls to assess non-specific binding

• HR functional assay controls:

  • Positive control: I-SceI transfection in DR-GFP reporter cells with intact HR

  • Negative control: DR-GFP reporter plasmid alone without I-SceI

  • BRCA1/2-depleted cells as established HR-deficient controls

  • RAD51 foci quantification as a parallel HR capacity readout

• Pharmacological intervention controls:

  • USP7 inhibition (P5091) to modulate CCDC6 stability

  • Concentration-dependent responses to establish dose-effect relationships

  • Time-course experiments to determine optimal treatment windows

  • Vehicle controls to account for solvent effects

• Rescue experiments:

  • Expression of wildtype CCDC6 in CCDC6-deficient cells

  • Expression of mutant CCDC6 (e.g., CCDC6T434A) to identify functional domains

  • Complementary approach using PARP inhibitor sensitivity as HR-deficiency readout

This control framework has been validated in studies showing that CCDC6 deficiency impairs HR repair, which can be rescued by re-expression of wildtype CCDC6 in otherwise deficient cells .

How can researchers accurately quantify and compare CCDC6 expression levels across different cell types and tissue samples?

For accurate quantification and comparison of CCDC6 expression across different samples:

• Multi-level expression analysis:

  • Protein level: Western blot with validated antibodies

  • mRNA level: Quantitative real-time PCR with appropriate reference genes

  • Post-translational regulation: Protein stability assays with cycloheximide chase

• Standardization procedures:

  • Use consistent lysis buffers with protease inhibitors

  • Normalize protein loading with multiple housekeeping controls (e.g., tubulin, PCNA)

  • Implement MIQE guidelines for qPCR experiments

  • Include common reference samples across independent experiments

• Quantification methods:

  • For western blots: Densitometric analysis with linear dynamic range validation

  • For IHC: H-score calculation (intensity × percentage positive cells)

  • For IF: Mean fluorescence intensity measurement with background subtraction

  • For qPCR: Relative quantification using the 2^-ΔΔCt method

• Reference panel development:
  Create a reference panel of cell lines with characterized CCDC6 expression:

  Cell Type	Protein Expression	mRNA Expression	Reference
  NTERA-2 (human EC)	High	High
  GC-1 (mouse spermatogonia)	Moderate	High
  GC-2 (mouse spermatocytes)	Low	Low
  TM4 (mouse Sertoli)	Low	Low
  MCF7 (human breast cancer)	High	Not reported
  HeLa (human cervical cancer)	High	Not reported

Research has shown that CCDC6 expression can vary significantly between tissue types and cell lines, with protein levels not always correlating with transcript abundance, suggesting post-translational regulation plays an important role in CCDC6 expression .

How can CCDC6 antibodies be utilized in developing predictive biomarkers for cancer therapy response?

CCDC6 antibodies can be implemented in predictive biomarker development through:

• Retrospective clinical sample analysis:

  • Perform CCDC6 immunohistochemistry on tumor tissue microarrays

  • Correlate expression patterns with treatment responses and outcomes

  • Establish cutoff values for "CCDC6-low" versus "CCDC6-high" tumors

  • Validate findings across multiple patient cohorts

• Companion diagnostic development:

  • Standardize staining protocols for clinical laboratory implementation

  • Develop scoring algorithms for pathologist interpretation

  • Compare manual versus digital quantification methods

  • Establish quality control procedures with reference standards

• Combined biomarker panels:
  Integrate CCDC6 with complementary markers for enhanced predictive value:

  Marker	Function	Combined Interpretation
  CCDC6	DNA repair regulator	Low expression indicates HR deficiency
  RAD51	HR effector protein	Reduced foci formation confirms HR defect
  γH2AX	DNA damage marker	Persistent foci indicate repair deficiency
  USP7	CCDC6 stabilizer	Low levels suggest post-translational regulation
  xCT/SLC7A11	Cystine transporter	High levels in CCDC6-low tumors indicate ROS tolerance

• Functional testing correlation:
  In studies of non-small cell lung cancer, low CCDC6 protein levels have been associated with cisplatin resistance but enhanced sensitivity to PARP inhibitors . This differential response pattern provides a foundation for biomarker-guided therapy selection.

Research has demonstrated that CCDC6 expression levels correlate with clinical outcomes, with low expression associated with lymph node metastasis and reduced survival in NSCLC patients . This evidence supports the development of CCDC6 as a clinically relevant biomarker.

What methodological approaches can resolve conflicting data between CCDC6 protein expression and mRNA levels in clinical samples?

When faced with discrepancies between CCDC6 protein and mRNA expression:

• Integrated multi-omics approach:

  • Perform parallel analyses of protein (IHC/western blot) and mRNA (qPCR/RNA-seq)

  • Add proteomic profiling to identify post-translational modifications

  • Include assessment of USP7 levels, a known regulator of CCDC6 stability

  • Analyze ubiquitination patterns in samples with low protein/high mRNA

• Technical validation:

  • Test multiple antibodies targeting different CCDC6 epitopes

  • Use multiple primer pairs spanning different exons for mRNA detection

  • Include positive and negative control samples with established expression patterns

  • Assess RNA and protein extraction efficiency from clinical material

• Alternative transcript analysis:

  • Screen for alternative splicing using exon-specific primers

  • Investigate 3'UTR variations that might affect mRNA stability

  • Assess for gene fusions involving CCDC6 (known in thyroid and lung cancers)

  • Quantify protein-coding potential of detected transcripts

• Regulatory mechanism investigation:

  • Measure half-life of CCDC6 protein in representative cell models

  • Assess microRNA profiles that may affect CCDC6 translation efficiency

  • Examine methylation status of the CCDC6 promoter

  • Investigate post-translational modifications affecting antibody recognition

Research has shown that in certain cell types (GC-2 spermatocytes and TM4 Sertoli cells), CCDC6 protein levels are nearly undetectable despite the presence of transcripts, suggesting that post-translational mechanisms rather than transcriptional regulation may control CCDC6 expression in these contexts .

How can researchers design experiments to evaluate CCDC6's role in determining sensitivity to PARP inhibitors versus conventional chemotherapy?

To investigate CCDC6's role in therapeutic response determination:

• In vitro sensitivity profiling:

  • Generate isogenic cell lines with CCDC6 knockout/knockdown and matched controls

  • Perform dose-response assays with PARP inhibitors (e.g., olaparib) and conventional agents (e.g., cisplatin)

  • Calculate IC50 values and combination indices

  • Assess cell death mechanisms through flow cytometry and biochemical assays

• Mechanistic investigation:

  • Quantify DNA damage accumulation via γH2AX immunofluorescence

  • Measure HR efficiency using DR-GFP reporter assays

  • Assess RAD51 foci formation following DNA damage

  • Evaluate PARP trapping on chromatin in CCDC6-deficient versus proficient cells

• Combination therapy optimization:

  • Test drug scheduling effects (concurrent versus sequential administration)

  • Determine optimal dosing ratios through isobologram analysis

  • Identify synergistic versus antagonistic interactions

  • Evaluate normal tissue toxicity in parallel

• In vivo validation:

  • Establish xenograft models with CCDC6-manipulated cells

  • Administer single agents and combinations at tolerable doses

  • Monitor tumor growth, survival, and pharmacodynamic markers

  • Correlate treatment outcomes with CCDC6 expression in tumor tissue

Research has demonstrated that CCDC6 attenuation confers resistance to cisplatin but sensitizes non-small cell lung cancer cells to PARP inhibitors . The combination of the two drugs has shown greater efficacy than either agent individually, with a combination index <1 indicating synergy. This differential response pattern provides a foundation for CCDC6-based treatment stratification strategies.

What are the critical factors affecting reproducibility when using CCDC6 antibodies across different experimental platforms?

To ensure reproducibility with CCDC6 antibodies across platforms:

• Antibody selection and validation:

  • Choose antibodies validated for specific applications (WB, IHC-P, IF/ICC)

  • Verify epitope location relative to known functional domains (aa 55-222 or aa 300-450)

  • Document lot-to-lot variation through consistent positive controls

  • Consider polyclonal versus monoclonal antibodies for different applications

• Sample preparation optimization:

  • For western blot: Include proteasome inhibitors during lysis

  • For IHC-P: Standardize fixation time and antigen retrieval methods

  • For IF/ICC: Optimize permeabilization conditions for cytoskeletal detection

  • For all applications: Use freshly prepared samples when possible

• Protocol standardization:

  Parameter	Western Blot	IHC-P	IF/ICC
  Recommended dilution	1:500-1:2000	1:50-1:200	1:50-1:200
  Blocking solution	5% BSA in TBST	1-5% normal serum	1-5% BSA in PBS
  Incubation time	Overnight, 4°C	1-2 hours, RT or overnight, 4°C	1-2 hours, RT
  Detection system	HRP/ECL	DAB/AP	Fluorescent secondary
  Positive controls	MCF7, HeLa, NTERA-2	Human testis, thyroid	HeLa, MCF7

• Data acquisition standardization:

  • Use consistent exposure times for western blot imaging

  • Standardize microscope settings for IHC and IF quantification

  • Implement automated analysis algorithms to reduce observer bias

  • Include technical and biological replicates for statistical validation

Research has shown that CCDC6 detection can be affected by protein stability issues, with USP7 inhibition significantly reducing CCDC6 half-life . Additionally, observed molecular weights may vary (56kDa/70kDa observed vs. 53kDa calculated) due to post-translational modifications , requiring careful consideration during data interpretation.

How can researchers overcome challenges in detecting low CCDC6 expression levels in clinical samples?

For enhanced detection of low CCDC6 expression in clinical samples:

• Signal amplification techniques:

  • For IHC: Implement tyramide signal amplification (TSA) systems

  • For western blot: Use high-sensitivity ECL substrates or fluorescent detection

  • For IF: Employ quantum dot conjugates or amplification kits

  • For all techniques: Optimize primary antibody concentration and incubation time

• Sample enrichment approaches:

  • Perform laser capture microdissection to isolate specific cell populations

  • Enrich for nuclear versus cytoplasmic fractions during protein extraction

  • Use phospho-enrichment techniques if targeting phosphorylated CCDC6 forms

  • Consider immunoprecipitation before western blotting for concentrated detection

• Alternative detection methodologies:

  • Proximity ligation assay (PLA) for in situ protein interaction visualization

  • Nanostring technology for simultaneous protein-mRNA detection

  • Mass spectrometry-based targeted proteomics for absolute quantification

  • Digital protein analysis platforms for single-molecule sensitivity

• Technical optimization for clinical samples:

  • Minimize cold ischemia time during tissue collection

  • Standardize fixation protocols for consistent epitope preservation

  • Optimize antigen retrieval methods for formalin-fixed tissues

  • Use automated staining platforms for consistency across samples

Research has demonstrated that CCDC6 expression varies significantly across tissue types, with some cell populations naturally expressing low levels that require enhanced detection methods . Additionally, approximately 30% of NSCLC tumors express low levels of CCDC6 , making sensitive detection methodologies crucial for accurate patient stratification.