CCDC181 Antibody

What is CCDC181 and what cellular functions does it serve?

CCDC181 (Coiled-Coil Domain Containing 181, also known as C1orf114) is a microtubule-binding protein that plays essential roles in sperm flagellum biogenesis and male fertility. It is an evolutionarily conserved component of the flagellar proteome found across diverse species . CCDC181 localizes to the microtubular manchette of elongating spermatids and to the sperm flagella . Expression analysis reveals that CCDC181 is predominantly expressed in male germ cells, with expression increasing during the haploid phase of spermatogenesis . While its primary function appears in reproductive biology, CCDC181 is also found in tissues containing ciliated epithelia, specifically localizing to the basal half of motile cilia, but notably absent in primary non-motile cilia . Recent research has identified CCDC181 as a microtubule-binding protein that interacts with Hook1, potentially serving as cargo for this microtubule-associated protein . Additionally, it may interact with protein phosphatase 1 (PP1) catalytic subunits, suggesting a role in mediating ciliary motility .

What are the key specifications of commercially available CCDC181 antibodies?

Several commercial sources offer CCDC181 antibodies with varying specifications important for research applications. The table below summarizes key characteristics of currently available antibodies:

Most commercially available CCDC181 antibodies are rabbit polyclonal antibodies with demonstrated reactivity against human CCDC181 protein . While all are suitable for immunohistochemistry applications, some have also been validated for Western blotting . The recommended dilution for immunohistochemistry is consistently 1:500-1:1000 across different manufacturers .

What are the recommended protocols for CCDC181 antibody validation?

Validating CCDC181 antibodies requires multiple complementary approaches to ensure specificity and sensitivity. Based on published research methodologies, the following protocol steps are recommended:

• Western Blot Validation: Use testis or sperm protein extracts alongside controls. Prepare protein lysates using lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, pH 7.5) containing protease inhibitor cocktail. After tissue grinding and ultrasonication, centrifuge lysates at 16,000 × g for 20 min at 4°C . The expected molecular weight for CCDC181 is approximately 60 kDa .

• Knockout Model Validation: Generate or obtain CCDC181 knockout models using CRISPR/Cas9 technology. Sample preparation can follow established protocols using gRNAs targeting exon 2 as demonstrated in published research (gRNA1: 5'-TGTCCCAAGTATTCAGCCGT-3' and gRNA2: 5'-GGATCCAACGGCTGAATACT-3') . This provides a negative control tissue to confirm antibody specificity.

• Immunohistochemistry Cross-Validation: Compare staining patterns across tissues known to express CCDC181 (testes, ciliated epithelia) with those that don't. Pay particular attention to subcellular localization patterns at the manchette of elongating spermatids and sperm flagella .

• Recombinant Protein Controls: Express tagged versions of CCDC181 in heterologous systems (e.g., HEK293T cells transfected with CCDC181-Flag) to serve as positive controls .

• Co-localization Studies: Perform dual-labeling with established markers of structures where CCDC181 localizes (manchette, flagella, cilia) to confirm expected spatial distribution patterns.

What is the optimal protocol for immunohistochemistry using CCDC181 antibodies?

For optimal immunohistochemistry results with CCDC181 antibodies, the following protocol is recommended based on published research methodologies:

• Sample Preparation: Fix tissue samples in 4% paraformaldehyde and embed in paraffin or prepare frozen sections depending on your experimental needs.

• Antigen Retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block non-specific binding with 5% normal serum (matching the species of secondary antibody) in PBS with 0.1% Triton X-100 for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute CCDC181 antibody at 1:500-1:1000 in blocking solution and incubate sections overnight at 4°C . This dilution range has been consistently recommended across different commercial antibodies.

• Secondary Antibody: Use appropriate HRP-conjugated or fluorescently-labeled secondary antibodies at manufacturer's recommended dilutions (typically 1:200-1:1000).

• Detection: For chromogenic detection, use DAB or similar substrate. For fluorescence, use appropriate fluorophore-conjugated secondary antibodies.

• Controls: Always include a negative control (primary antibody omitted) and, if possible, tissue from CCDC181 knockout models .

• Counterstaining: Use hematoxylin for brightfield microscopy or DAPI for nuclear counterstaining in fluorescence applications.

When analyzing results, pay particular attention to expression in ciliated tissues and male reproductive tissues, especially at subcellular structures like the manchette and flagella .

How can CCDC181 antibodies be applied in studying male infertility models?

CCDC181 antibodies serve as powerful tools for investigating male infertility, particularly cases involving sperm flagellar abnormalities. Recent research has established CCDC181 as essential for sperm flagellum biogenesis and male fertility . When designing experiments to study male infertility models:

• MMAF Phenotype Characterization: CCDC181 knockout mice display characteristics consistent with Multiple Morphological Abnormalities of the Flagella (MMAF) syndrome, a condition characterized by severe sperm flagellar defects . CCDC181 antibodies can be used to assess protein localization in spermatozoa from infertile patients with suspected MMAF.

• Comparative Analysis Protocol: Collect sperm samples from control and experimental groups (patient samples or animal models). Fix in 4% paraformaldehyde, permeabilize with 0.5% Triton X-100, and perform immunofluorescence using CCDC181 antibody (1:500) alongside other flagellar markers. Compare expression patterns, paying particular attention to the midpiece localization.

• Genotype-Phenotype Correlation: When analyzing patient samples with potential CCDC181 mutations, combine genotyping with immunostaining to establish causality. CCDC181 antibodies can reveal whether specific mutations affect protein expression, stability, or localization.

• Interaction Partner Analysis: Use CCDC181 antibodies in co-immunoprecipitation experiments to investigate interactions with known partners like LRRC46, which has been shown to display similar MMAF phenotypes when disrupted . For co-IP, use testis lysates in cold immunoprecipitation buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 2.5 mM EDTA) with 1 mM PMSF, incubating with anti-CCDC181 antibody (1:200) overnight at 4°C .

• Rescue Experiments: In CCDC181-deficient models, immunostaining with CCDC181 antibodies can confirm successful protein re-expression in rescue experiments, providing valuable insights into structure-function relationships.

What is the relationship between CCDC181 and other flagellar/ciliary proteins in experimental design?

CCDC181's interactions with other flagellar and ciliary proteins must be considered when designing comprehensive studies of these complex structures. Current evidence suggests several important relationships:

• CCDC181-Hook1 Interaction: CCDC181 has been shown to interact with Hook1, a microtubule-binding protein also localized to the manchette of elongating spermatids . When designing experiments investigating manchette function, consider dual immunolabeling with both CCDC181 and Hook1 antibodies to assess co-localization. FRET analysis has verified this interaction, and yeast two-hybrid assays have identified specific interacting regions .

• CCDC181-LRRC46 Relationship: Immunoprecipitation-mass spectrometry and co-IP experiments have demonstrated that CCDC181 interacts with LRRC46, another MMAF-related protein . Both proteins accumulate at the midpiece of the spermatozoon tail. When designing experiments, assess LRRC46 localization in CCDC181-deficient models, as research has shown deficient LRRC46 signals along flagella in Ccdc181−/− mice .

• Protein Phosphatase 1 Interactions: CCDC181 has been identified as a putative interacting partner of different catalytic subunits of Protein Phosphatase 1 (PP1), including PP1γ2, which plays crucial roles in sperm motility . When studying phosphorylation-dependent regulation of flagellar function, consider how CCDC181 might mediate PP1 activity.

• Differential Expression Protocol: For comprehensive protein interaction studies, perform quantitative immunoblotting across developmental stages of spermatogenesis, comparing CCDC181 expression with known interaction partners. Correlate expression patterns with functional outcomes in knockout models.

• Tissue-Specific Differences: While CCDC181 is localized on motile cilia, knockout mice did not exhibit primary ciliary dyskinesia, suggesting tissue-specific functional redundancy . When designing experiments, consider tissue-specific differences in CCDC181 requirement, particularly between sperm flagella and respiratory cilia.

What technical challenges exist in distinguishing CCDC181 localization patterns across different cellular structures?

Accurate determination of CCDC181 localization presents several technical challenges that researchers must address through careful experimental design:

• Distinguishing Manchette vs. Flagellar Localization: CCDC181 localizes to both the microtubular manchette of elongating spermatids and to the flagella of mature sperm . To differentiate these localizations:

  • Use developmental staging of spermatogenesis to separate manchette-containing cells from those with developing flagella

  • Employ super-resolution microscopy techniques (STED, STORM) to resolve close but distinct microtubular structures

  • Perform co-localization with manchette-specific markers (e.g., Hook1) and flagella-specific markers

• Motile vs. Non-motile Cilia Discrimination: CCDC181 is found in the basal half of motile cilia but not in primary non-motile cilia . This distinction requires:

  • Careful tissue selection (respiratory epithelium for motile cilia vs. kidney tubules for primary cilia)

  • Co-staining with acetylated tubulin (general ciliary marker) alongside motile cilia-specific markers (DNAH5, RSPH4A)

  • Z-stack confocal microscopy to precisely determine localization within the ciliary axoneme

• Antibody Penetration Challenges: Dense microtubular structures like flagella may have restricted antibody accessibility, requiring:

  • Extended permeabilization times (increase Triton X-100 to 1% for 30 minutes)

  • Antigen retrieval optimization specifically for microtubular structures

  • Comparison of different fixation methods (paraformaldehyde vs. methanol) for optimal epitope exposure

• Background Reduction Protocol: To minimize non-specific background that can obscure specific localization:

  • Increase blocking time to 2 hours with 10% serum

  • Add 1% BSA to antibody dilution buffer

  • Consider tyramide signal amplification for weak signals while maintaining specificity

  • Use knockout tissues as negative controls to establish baseline background levels

• Quantitative Assessment: Develop quantitative approaches to measure relative CCDC181 intensities across subcellular structures using line-scan analysis or region-of-interest intensity measurements in imaging software.

How can CCDC181 antibodies be utilized in genome-editing validation experiments?

When conducting CRISPR/Cas9 or other genome editing experiments targeting CCDC181, antibodies play a crucial role in validating successful gene modification at the protein level:

• Knockout Verification Protocol: For CCDC181 knockout models, the following validation steps utilizing antibodies are essential:

  • Western blot analysis of testis extracts from wild-type vs. knockout animals using anti-CCDC181 antibody at 1:500 dilution

  • Immunohistochemistry on testis sections to confirm absence of protein in knockout tissues

  • Immunofluorescence on sperm to verify loss of flagellar and manchette localization

• Mutation-Specific Detection: For point mutations or domain-specific modifications:

  • If the mutation affects the epitope recognized by the antibody, differential staining patterns between wild-type and mutant tissues can confirm successful editing

  • For mutations outside the antibody epitope, use immunoprecipitation followed by mass spectrometry to confirm the presence of the mutated protein

• Phenotype Analysis Pipeline: Combine CCDC181 immunostaining with functional and structural analyses:

  • Assess sperm morphology using phase-contrast microscopy

  • Evaluate sperm motility parameters

  • Perform transmission electron microscopy of flagellar ultrastructure

  • Correlate these findings with CCDC181 expression patterns

• Developmental Impact Assessment: Use CCDC181 antibodies to track protein expression throughout spermatogenesis in edited models:

  • Perform stage-specific immunostaining of seminiferous tubules

  • Quantify expression levels at different developmental stages

  • Compare timing of developmental defects with normal CCDC181 expression patterns

• Rescue Experiment Design: When conducting rescue experiments in CCDC181-deficient models:

  • Use epitope-tagged rescue constructs to distinguish endogenous from exogenous protein

  • Perform co-immunostaining with CCDC181 antibody and tag-specific antibodies

  • Assess restoration of normal localization patterns and interaction with partner proteins

The published protocol for generating Ccdc181 knockout mice using CRISPR/Cas9 targeting exon 2 with specific gRNAs (gRNA1: 5'-TGTCCCAAGTATTCAGCCGT-3' and gRNA2: 5'-GGATCCAACGGCTGAATACT-3') provides a validated approach for creating models for antibody validation .

What methodological approaches can resolve contradictory CCDC181 localization data across studies?

Researchers occasionally encounter contradictory data regarding CCDC181 localization. The following methodological approaches can help resolve these discrepancies:

• Antibody Epitope Mapping: Different antibodies may recognize distinct epitopes of CCDC181, potentially explaining discrepant localization patterns. Perform epitope mapping by:

  • Using multiple antibodies targeting different regions of CCDC181

  • Creating a panel of truncated CCDC181 constructs to identify regions recognized by each antibody

  • Conducting peptide competition assays to confirm epitope specificity

• Fixation-Dependent Artifact Assessment: Some localization patterns may be fixation-dependent artifacts. Compare:

  • Paraformaldehyde fixation (preserves protein-protein interactions)

  • Methanol fixation (better for some microtubular structures)

  • Glutaraldehyde fixation (improved ultrastructural preservation)

  • Live-cell imaging with fluorescently tagged CCDC181 to avoid fixation artifacts entirely

• Cross-Species Validation Protocol: Evolutionary conservation of CCDC181 allows cross-species validation:

  • Compare localization patterns in mouse, human, and other model organisms

  • Align protein sequences to identify conserved domains that might explain species-specific differences

  • Use species-specific knockout models to confirm antibody specificity across species

• Sample Preparation Standardization: Develop a standardized sample preparation protocol:

  • Precise timing of fixation post-sample collection

  • Consistent permeabilization conditions

  • Standardized blocking reagents

  • Identical antibody concentrations and incubation times

  • Controlled imaging parameters across studies

• Quantitative Colocalization Analysis: Apply rigorous quantitative methods:

  • Calculate Pearson's correlation coefficients for colocalization with known markers

  • Perform line-scan analysis across cellular structures

  • Use super-resolution microscopy (STED, STORM) for nanoscale localization precision

  • Apply deconvolution algorithms to improve spatial resolution

How should experiments be designed to investigate CCDC181's role in ciliopathies?

Although CCDC181 knockout mice did not exhibit primary ciliary dyskinesia , the protein's conservation in ciliary proteomes suggests potential roles in human ciliopathies. The following experimental design considerations are important:

• Patient Cohort Selection Strategy: When screening ciliopathy patients for CCDC181 involvement:

  • Prioritize patients with unexplained male infertility, particularly MMAF phenotypes

  • Include patients with combined reproductive and respiratory phenotypes

  • Consider ciliopathy patients with normal cilia ultrastructure but impaired motility

• Tissue-Specific Expression Analysis: CCDC181 may have different functions across ciliated tissues:

  • Use quantitative RT-PCR to assess relative expression levels across tissues

  • Perform Western blotting with CCDC181 antibodies on protein extracts from various ciliated epithelia

  • Conduct immunohistochemistry on tissue microarrays containing multiple ciliated tissues

• Functional Redundancy Assessment: The lack of respiratory phenotypes in Ccdc181−/− mice suggests functional redundancy :

  • Identify and characterize CCDC181 paralogs or functionally similar proteins

  • Perform double-knockout experiments targeting CCDC181 and potential compensatory proteins

  • Use RNA-seq to identify upregulated genes in CCDC181-deficient ciliated tissues

• Ciliary Motility Analysis Protocol:

  • High-speed videomicroscopy (200+ frames per second) of ciliated epithelia

  • Quantification of ciliary beat frequency and waveform characteristics

  • Correlation of motility parameters with CCDC181 expression levels

  • Transmission electron microscopy of ciliary ultrastructure

• Protein-Protein Interaction Network: CCDC181's interaction with PP1 suggests roles in phosphorylation-dependent regulation of ciliary motility :

  • Use proximity labeling methods (BioID, APEX) with CCDC181 as bait to identify cilium-specific interactors

  • Perform phosphoproteomic analysis comparing wild-type and CCDC181-deficient cilia

  • Identify ciliary substrates of PP1 that might be regulated through CCDC181