CCDC57 Antibody

What is CCDC57 and what biological functions has it been associated with?

CCDC57 (Coiled-coil domain-containing protein 57) is a multifunctional protein that plays critical roles in several cellular processes. Recent research has revealed its significance in motile cilia function, particularly in the coupling of basal foot (BF) and axonemal orientation (AO) necessary for coordinated ciliary beating across epithelial tissues . The protein has also been implicated in centriole duplication, mitotic processes, and ciliogenesis . Evolutionary analysis suggests that CCDC57's presence correlates with epithelial motile cilia development, indicating its conserved role in ciliary function across species . In knockout models, CCDC57 deficiency leads to severe phenotypes including hydrocephalus due to impaired cerebrospinal fluid flow, demonstrating its essential function in maintaining proper ciliary movement and subsequent physiological processes .

What types of CCDC57 antibodies are available for research applications?

Multiple validated antibodies targeting different epitopes of CCDC57 are available for research applications. The most well-characterized include:

• Anti-CCDC57 antibody HPA023326 - A rabbit polyclonal antibody that targets the immunogen sequence QEEEVKLKAQVARSQQDIERYKQQLSLAVERERSLERDQVQLGLDWQRRCDDIERDQIQKSEALIQGLSMAKSQVAAKL .

• Anti-CCDC57 antibody HPA023342 - A rabbit polyclonal antibody targeting a different epitope with the immunogen sequence AQAGQAGGSVQAGQAGGSVQAGPVSSGLALRKLGDRVQLLNLLVTRLRQKVLREPLEPAALQRELPREVDQVHLEVLELRKQVAELGKHLRIAQHGGAEPSGRKQPPASDAVALGREVGA .

Both antibodies are affinity-isolated and provided in buffered aqueous glycerol solutions, designed specifically for human CCDC57 protein detection .

How do researchers validate the specificity of CCDC57 antibodies?

Validation of CCDC57 antibodies involves multiple rigorous approaches:

• Protein Array Testing: Commercial antibodies undergo screening against arrays containing 364 human recombinant protein fragments to assess cross-reactivity and specificity .

• Tissue Microarray Analysis: Comprehensive testing on tissue arrays comprising 44 normal human tissues and 20 common cancer type tissues confirms appropriate expression patterns and specificity in diverse cellular contexts .

• Western Blot Validation: For antibodies appropriate for immunoblotting (like HPA023342), molecular weight verification and band specificity are assessed across multiple cell and tissue lysates .

• Knockout/Knockdown Controls: Advanced validation includes testing in CCDC57-deficient models to confirm signal absence, which represents the gold standard for antibody specificity .

• Subcellular Localization Correlation: Immunofluorescence patterns are compared with known localization data from the Human Protein Atlas and functional studies examining centriolar and ciliary structures .

What are the recommended protocols for using CCDC57 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of CCDC57 in formalin-fixed, paraffin-embedded tissues:

• Tissue Preparation:

  • Standard fixation in 10% neutral buffered formalin for 24-48 hours

  • Paraffin embedding and sectioning at 4-6 μm thickness

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooking for 15-20 minutes or microwave heating for 20 minutes

• Immunostaining Protocol:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide (10 minutes)

  • Block non-specific binding with 5% normal serum (30-60 minutes)

  • Apply primary antibody at dilutions of 1:200-1:500 at 4°C overnight

  • Incubate with appropriate secondary antibody and detection system

  • Counterstain with hematoxylin to visualize nuclei

• Quality Controls:

  • Include positive control tissues with known CCDC57 expression

  • Include negative controls by omitting primary antibody

  • Consider parallel staining with both HPA023326 and HPA023342 antibodies to confirm specificity through epitope validation

Specific attention should be paid to epithelial tissues with motile cilia, such as respiratory epithelium and ependymal cells, which demonstrate strong CCDC57 expression patterns .

How should researchers optimize Western blot protocols for CCDC57 detection?

For optimized Western blot detection of CCDC57:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for protein extraction

  • Include phosphatase inhibitors if studying potential post-translational modifications

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Gel Electrophoresis and Transfer:

  • Load 20-40 μg of total protein per lane

  • Use 8-10% SDS-PAGE gels due to CCDC57's molecular weight

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• Immunoblotting:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with HPA023342 antibody at 0.04-0.4 μg/mL concentration (approximately 1:1000-1:10000 dilution)

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000)

  • Develop using enhanced chemiluminescence reagents

• Expected Results:

  • CCDC57 typically appears as a band at approximately 108-115 kDa

  • In some cell types with post-translational modifications, additional bands may be observed

• Validation Controls:

  • Include loading controls (β-actin, GAPDH)

  • Consider including lysates from tissues known to express high levels of CCDC57, such as ciliated epithelium

What experimental approaches can be used to study CCDC57's role in ciliary function?

Based on recent research findings, several methodologies can effectively investigate CCDC57's ciliary functions:

• Live Imaging of Ciliary Beating:

  • High-speed videomicroscopy (200-500 frames per second) to capture ciliary beat patterns

  • Differential interference contrast (DIC) microscopy for enhanced visualization

  • Fluorescent labeling of ciliary components combined with confocal microscopy for spatial relationships

• Basal Body and Basal Foot Orientation Analysis:

  • Transmission electron microscopy to visualize basal foot orientation

  • Immunofluorescence microscopy using antibodies against basal body markers (γ-tubulin) and basal foot components

  • Quantification of orientation angles to assess planar cell polarity

• Functional Flow Assays:

  • Bead tracking to measure fluid flow generated by motile cilia

  • Particle image velocimetry to analyze flow patterns and velocities

  • In vitro airway epithelial models to assess mucociliary clearance

• Genetic Manipulation Strategies:

  • CRISPR/Cas9-mediated knockout or knockdown of CCDC57

  • Rescue experiments with wild-type or mutant CCDC57 constructs

  • Domain-specific mutations to identify functional regions

• Animal Models:

  • Analysis of CCDC57-deficient mice, particularly focusing on hydrocephalus development and respiratory function

  • Primary cell cultures from these models for detailed molecular analyses

These methodologies have revealed that CCDC57 is essential for coupling basal foot orientation with axonemal orientation, which is critical for coordinated, directional ciliary beating across multiciliated epithelia .

How can researchers differentiate between CCDC57's roles in centriole duplication versus ciliary orientation?

Distinguishing between CCDC57's multiple cellular functions requires specific experimental designs:

• Temporal Analysis:

  • Synchronized cell populations to examine protein dynamics throughout the cell cycle

  • Time-lapse imaging with fluorescently tagged CCDC57 to track localization changes

  • Conditional knockout systems that deplete CCDC57 at specific cell cycle stages

• Structure-Function Analysis:

  • Domain-specific mutations to identify regions responsible for centriole duplication versus ciliary functions

  • Expression of truncated CCDC57 constructs to determine minimal functional domains

  • Proximity labeling approaches (BioID, APEX) to identify distinct interactome networks associated with each function

• Differential Localization Studies:

  • Super-resolution microscopy to precisely map CCDC57 localization relative to centriolar and ciliary structures

  • Co-immunoprecipitation with stage-specific markers of centriole duplication versus basal body maturation

  • Fractionation experiments to separate centrosomal versus ciliary compartments

• Functional Readouts:

  • For centriole duplication: quantify centrosome numbers and mitotic spindle formation

  • For ciliary orientation: assess basal foot alignment and multiciliary beat coordination

  • Compare phenotypes using specific inhibitors of cell cycle progression versus ciliary assembly

Recent research indicates that while CCDC57 participates in both processes, its critical role in coupling basal foot and axonemal orientation represents a distinct function that specifically affects coordinated ciliary beating across epithelial tissues .

What are common technical challenges when using CCDC57 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with CCDC57 antibodies:

• Weak Signal Intensity:

  • Challenge: Low signal-to-noise ratio in immunostaining or Western blots

  • Solution: Optimize antigen retrieval (for IHC) by testing multiple buffers (citrate pH 6.0, EDTA pH 9.0); increase antibody concentration incrementally; extend primary antibody incubation to overnight at 4°C; use signal amplification systems like TSA (tyramide signal amplification)

• Background Staining:

  • Challenge: Non-specific binding creating high background

  • Solution: Increase blocking time (2-3 hours); use stronger blocking agents (5% BSA or 10% normal serum); add 0.1-0.3% Triton X-100 for membrane permeabilization; perform more extensive washing steps; consider using monovalent Fab fragments to block endogenous immunoglobulins in tissue sections

• Epitope Accessibility Issues:

  • Challenge: Masked epitopes, particularly in highly structured ciliary components

  • Solution: Test multiple fixation protocols (PFA versus methanol); compare different antigen retrieval methods; consider using alternative antibodies targeting different epitopes (HPA023326 versus HPA023342)

• Specificity Concerns:

  • Challenge: Cross-reactivity with related coiled-coil domain proteins

  • Solution: Validate with knockout/knockdown controls; perform peptide competition assays; use both antibodies in parallel to confirm consistent staining patterns; consider pre-absorption with potential cross-reactive proteins

• Variable Results Across Applications:

  • Challenge: Antibody performs well in IHC but poorly in IP or IF

  • Solution: Optimize application-specific protocols; consider using HPA023342 for Western blotting applications and HPA023326 for IHC ; adjust fixation and permeabilization conditions for immunofluorescence

How do researchers reconcile contradictory findings regarding CCDC57 subcellular localization and function?

When faced with contradictory findings about CCDC57:

• Methodological Differences Assessment:

  • Compare fixation methods, as CCDC57 localization can vary significantly between PFA and methanol fixation

  • Evaluate antibody specificity through knockout controls and epitope mapping

  • Consider differences in cell types and developmental stages used across studies

• Functional Domain Analysis:

  • CCDC57 contains multiple functional domains that may localize to different subcellular structures

  • Different antibodies (HPA023326 vs. HPA023342) target distinct epitopes, potentially revealing different aspects of CCDC57 localization

  • Generate domain-specific antibodies or tagged constructs to track specific protein regions

• Dynamic Localization Possibilities:

  • CCDC57 may shuttle between multiple cellular locations depending on cell cycle stage or differentiation state

  • Live-cell imaging with fluorescently tagged CCDC57 can reveal temporal dynamics

  • Synchronization experiments can determine cell-cycle dependent localization patterns

• Context-Dependent Functions:

  • The initial speculation about CCDC57's function has evolved with new research findings

  • Tissue-specific interactors may influence CCDC57's function and localization

  • Compare results across multiple model systems (cell lines, primary cells, animal models)

• Experimental Validation Approaches:

  • Perform rescue experiments with wild-type CCDC57 in knockout systems

  • Use proximity labeling methods to identify location-specific interaction partners

  • Implement functional assays relevant to each proposed function (centriole duplication, ciliary orientation, etc.)

The recent finding that CCDC57 mediates coupling between basal foot and axonemal orientation represents a significant advance in understanding its function, contradicting earlier, more limited hypotheses about its role .

How is CCDC57 implicated in pathological conditions, and what research models are appropriate for studying these connections?

CCDC57 dysfunction has been linked to several pathological conditions, which can be studied using specific research models:

• Hydrocephalus:

  • Pathological Connection: CCDC57-deficient mice develop severe hydrocephalus due to impaired cerebrospinal fluid flow resulting from dysfunctional ependymal cilia

  • Research Models:

    • CCDC57 knockout mice for in vivo studies of ventricular enlargement progression

    • Primary ependymal cell cultures for detailed ciliary beat frequency analysis

    • Human patient-derived cells for translational relevance

• Respiratory Disorders:

  • Pathological Connection: CCDC57's role in tracheal multicilia alignment suggests potential involvement in respiratory pathologies similar to primary ciliary dyskinesia

  • Research Models:

    • Air-liquid interface cultures of human bronchial epithelial cells

    • Ex vivo tracheal explants for mucociliary clearance assessment

    • Mouse models for integrated respiratory function evaluation

• Reproductive System Dysfunction:

  • Pathological Connection: Given CCDC57's role in motile cilia, potential implications exist for fertility issues related to fallopian tube ciliary function or sperm flagellar movement

  • Research Models:

    • Oviduct epithelial cultures for ciliary function assessment

    • Sperm motility analyses in CCDC57-deficient models

    • Fertility studies in animal models with tissue-specific CCDC57 deletion

• Neurodevelopmental Disorders:

  • Pathological Connection: The severe consequences of ependymal ciliary dysfunction suggest potential roles in neurodevelopmental processes

  • Research Models:

    • Neural organoids to model early developmental impacts

    • Conditional knockout mouse models with temporal control of CCDC57 deletion

    • Cerebrospinal fluid flow dynamics studies using contrast-enhanced MRI

When studying these conditions, researchers should implement comprehensive phenotyping approaches that connect molecular defects to tissue-level dysfunction and ultimately to organismal pathology .

What quantitative methods can researchers use to assess CCDC57-dependent ciliary orientation and coordination?

Several quantitative methods can effectively measure CCDC57-dependent ciliary functions:

• Basal Foot Orientation Analysis:

  • Method: Transmission electron microscopy of basal bodies with angular measurement

  • Quantification: Calculate the vector of basal foot orientation relative to tissue axis

  • Statistical Analysis: Circular statistics to determine mean orientation and variance

  • Expected Results: CCDC57-deficient tissues show random basal foot orientations compared to the coordinated alignment in wild-type samples

• Ciliary Beat Pattern Analysis:

  • Method: High-speed videomicroscopy (200-500 fps) of ciliated cells

  • Quantification:

    • Beat frequency (Hz) measurement

    • Power spectrum analysis of beating patterns

    • Effective stroke direction relative to tissue axis

  • Statistical Analysis: Comparison of directional consistency between adjacent cells

  • Expected Results: CCDC57-deficient cilia maintain beating ability but lack coordinated directionality across the epithelium

• Flow Velocity Mapping:

  • Method: Fluorescent microbead tracking over ciliated surfaces

  • Quantification:

    • Vector field generation of flow directions and magnitudes

    • Calculation of flow coherence across the field

  • Statistical Analysis: Spatial autocorrelation of flow vectors

  • Expected Results: Reduced net flow and increased turbulence in CCDC57-deficient tissues

• Axonemal Orientation Assessment:

  • Method: Immunofluorescence of ciliary markers with confocal microscopy

  • Quantification:

    • Angular measurement of central pair microtubule orientation

    • Correlation between basal body and axonemal orientations

  • Statistical Analysis: Linear regression analysis of correlation coefficients

  • Expected Results: CCDC57 deficiency leads to uncoupling between basal foot orientation and axonemal orientation

These quantitative approaches collectively demonstrate that CCDC57 functions specifically in the coupling mechanism between basal foot orientation and axonemal orientation, a critical step in establishing coordinated ciliary beating across epithelial surfaces .

What emerging technologies might advance our understanding of CCDC57's molecular mechanisms?

Several cutting-edge technologies show promise for elucidating CCDC57's precise molecular functions:

• Cryo-Electron Tomography:

  • Application: Visualize CCDC57's native structure at the basal body-axoneme interface at near-atomic resolution

  • Advantage: Reveals structural changes in CCDC57-deficient cilia without artifacts from traditional EM preparation

  • Expected Insights: Identification of the physical links that couple basal foot structures to axonemal components

• Proximity-dependent Biotinylation (BioID/TurboID):

  • Application: Map CCDC57's dynamic interactome at different subcellular locations

  • Advantage: Captures even transient interactions in living cells

  • Expected Insights: Identify different protein complexes associated with CCDC57's centriolar versus ciliary functions

• Live Super-resolution Microscopy:

  • Application: Track CCDC57 dynamics during ciliogenesis and basal body orientation

  • Advantage: Combines nanometer resolution with temporal information

  • Expected Insights: Visualize the real-time reorientation of basal bodies and the recruitment of CCDC57

• Single-cell Transcriptomics and Proteomics:

  • Application: Profile expression patterns in CCDC57-deficient versus wild-type multiciliated cells

  • Advantage: Reveals compensatory pathways and secondary effects

  • Expected Insights: Identify gene regulatory networks controlled by or controlling CCDC57 expression

• CRISPR Base Editing for Structure-Function Analysis:

  • Application: Create precise amino acid substitutions to map functional domains

  • Advantage: More subtle than knockout approaches, allowing identification of specific interaction interfaces

  • Expected Insights: Determine which CCDC57 domains mediate basal foot coupling versus other functions

These technologies will help resolve the apparent contradictions in earlier research regarding CCDC57's function, building upon the recent discovery of its essential role in coupling basal foot orientation to axonemal orientation for coordinated ciliary beating .

How might understanding CCDC57 function contribute to therapeutic approaches for ciliopathies?

Research into CCDC57 has significant therapeutic implications for ciliopathies:

• Diagnostic Applications:

  • Development of CCDC57 antibody-based diagnostics for identifying subtypes of ciliopathies

  • Implementation of targeted genetic testing for CCDC57 mutations in patients with hydrocephalus and respiratory symptoms

  • Creation of functional assays measuring ciliary orientation as diagnostic tools

• Gene Therapy Approaches:

  • Design of AAV-based vectors for CCDC57 delivery to affected tissues

  • Development of tissue-specific promoters for targeted expression in ciliated epithelia

  • Testing of mRNA-based therapies for transient CCDC57 expression

• Small Molecule Interventions:

  • High-throughput screening for compounds that stabilize basal foot-axoneme coupling

  • Identification of drugs that can compensate for CCDC57 deficiency by strengthening parallel pathways

  • Development of treatments that address downstream consequences of ciliary misalignment

• Cell-based Therapies:

  • Engineering of ciliated epithelial cells with enhanced CCDC57 expression

  • Development of stem cell-derived multiciliated cells for transplantation

  • Testing of extracellular vesicle delivery of CCDC57 mRNA or protein

• Precision Medicine Strategies:

  • Classification of ciliopathy patients based on CCDC57 mutation status

  • Tailoring of interventions based on specific mechanisms of CCDC57 dysfunction

  • Combination therapies addressing both CCDC57 deficiency and secondary pathological processes

The recent findings demonstrating CCDC57's essential role in coordinating ciliary orientation across epithelia provides a crucial mechanistic foundation for these therapeutic approaches, particularly for conditions involving ependymal cilia dysfunction and subsequent hydrocephalus .