ccdc103 Antibody

What is CCDC103 and why is it important for research?

CCDC103 is an oligomeric coiled-coil domain protein that specifically binds and stabilizes polymerized microtubules, playing a critical role in dynein arms assembly . Recent research has demonstrated that CCDC103 has conserved expression in vertebrate myeloid lineages, including primitive macrophages and neutrophils, and localizes with cytoplasmic dynein (DYNH1C1) on microtubules within their cytoplasm . Mutations in CCDC103 are associated with Primary Ciliary Dyskinesia (PCD), making it an important target for studying both ciliary function and extraciliary processes such as myeloid cell migration and proliferation . Studies in zebrafish mutants have shown that myeloid cells lacking CCDC103 exhibit decreased proliferation, disrupted directed migration to wound sites, and abnormal morphology, consistent with loss of cytoplasmic microtubule stability .

How do I select the appropriate CCDC103 antibody for my experimental needs?

Selecting the appropriate CCDC103 antibody depends on your experimental design, target species, and application requirements:

• Target specificity: Consider which domain or epitope of CCDC103 you need to target. Available antibodies include those targeting specific amino acid regions (e.g., AA 1-242, AA 160-209, C-terminal) .

• Species reactivity: Verify cross-reactivity with your experimental model. Some antibodies react only with human CCDC103, while others cross-react with mouse, rat, cow, dog, horse, or rabbit proteins .

• Application compatibility: Match the antibody to your intended application. Available antibodies are optimized for various techniques including ELISA, Western blotting (WB), and immunohistochemistry (IHC) .

• Conjugation requirements: Determine if you need an unconjugated antibody or one conjugated to a detection molecule (Biotin, HRP, FITC) based on your detection method .

• Clonality: Most available CCDC103 antibodies are polyclonal, derived from rabbit hosts, which may offer advantages in detecting native proteins .

What are the key domains of CCDC103 that antibodies typically recognize?

CCDC103 antibodies are available that recognize several key domains:

• Coiled-coil domain (AA 1-242): This recognition site encompasses the full-length protein and includes the functional coiled-coil domains that facilitate protein-protein interactions and oligomerization .

• C-terminal domain: Antibodies targeting the C-terminus can be useful for detecting potential truncation mutations, such as G128fs* that cause severe PCD phenotypes .

• Mid-region epitopes (AA 160-209): This region includes residues near the H154P mutation site, which has been implicated in both PCD and fertility disorders .

The choice of domain-specific antibody should align with your research questions, as mutations in different domains affect CCDC103 interactions with binding partners like DYNC1H1 and SPAG6 to varying degrees .

How can I optimize Western blotting protocols for CCDC103 detection?

For optimal Western blotting results with CCDC103 antibodies:

• Sample preparation:

  • Extract total protein from cells using a lysis buffer containing protease inhibitors

  • Include phosphatase inhibitors if investigating potential phosphorylation states

  • Process samples on ice to prevent protein degradation

• Gel electrophoresis optimization:

  • Use 10-12% polyacrylamide gels for optimal resolution of CCDC103 (~28 kDa)

  • Include positive controls from cells known to express CCDC103 (e.g., HL-60 cells or myeloid lineages)

• Transfer and detection:

  • Perform semi-dry or wet transfer at 100V for 1 hour or 30V overnight

  • Block with 5% non-fat milk or BSA depending on antibody specifications

  • Use antibody dilutions as recommended (typically 1:500 to 1:2000)

  • If signal is weak, consider using enhanced chemiluminescence substrates

• Controls:

  • Include samples from known CCDC103-expressing tissues

  • Consider using recombinant CCDC103 protein as a positive control

  • For negative controls, use tissues from CCDC103 knockout models or siRNA-treated cells

Research indicates that CCDC103 localizes differently in various myeloid cell types, showing larger, sparse puncta in undifferentiated cells versus a more diffuse distribution in differentiated cells . These localization patterns may affect extraction efficiency and detection sensitivity.

What immunofluorescence protocols best reveal CCDC103 subcellular localization?

For optimal visualization of CCDC103 subcellular localization by immunofluorescence:

• Fixation method selection:

  • Use 4% paraformaldehyde for 10-15 minutes at room temperature

  • For better preservation of microtubule structures, consider methanol fixation (-20°C for 5-10 minutes)

• Permeabilization optimization:

  • For cytoplasmic CCDC103, use 0.1-0.2% Triton X-100

  • For preservation of microtubule associations, consider mild detergents like 0.05% saponin

• Co-staining recommendations:

  • Include α-Tubulin staining to visualize microtubule networks

  • Co-stain with DYNC1H1 antibodies to detect co-localization at microtubule organizing centers (MTOCs)

  • For myeloid cells, include lineage markers (CD14 for macrophages, CD15 for neutrophils)

• Cell-type specific considerations:

  • For undifferentiated myeloid progenitor cells, focus on perinuclear regions and MTOCs

  • In differentiated neutrophils and macrophages, look for smaller punctate and diffuse distribution patterns

Studies have demonstrated that CCDC103 localization patterns differ between cell types, with perinuclear localization in primitive neutrophils and association with the cytoplasmic microtubule network in various myeloid cells .

How can I assess CCDC103 expression patterns in different cell lineages?

To effectively assess CCDC103 expression across different cell lineages:

• RT-PCR/qPCR approach:

  • Design primers spanning exon junctions to avoid genomic DNA amplification

  • Normalize expression to stable housekeeping genes (GAPDH, β-actin)

  • For myeloid lineages, consider flow-sorting cells (e.g., using spi1b:EGFP+ markers in zebrafish)

• Flow cytometry method:

  • Perform intracellular staining with permeabilization using 0.1% saponin

  • Use conjugated CCDC103 antibodies (FITC or PE) for direct detection

  • Include appropriate isotype controls

• Single-cell analysis:

  • Consider scRNA-seq to profile CCDC103 expression across heterogeneous populations

  • For protein-level analysis, use mass cytometry (CyTOF) with metal-conjugated antibodies

• Hematopoietic differentiation models:

  • In zebrafish, co-inject mRNA encoding master hematopoietic regulators (scl/tal1 and lmo2) to expand the anterior CCDC103 expression domain

  • In human systems, analyze expression in CD34+/CD38- cells from whole blood and in HL-60 cell differentiation models

Research has shown that CCDC103 is expressed in both zebrafish and human myeloid cells, including CD34+/CD38- progenitors, whole cord blood, and the HL-60 promyelocytic leukemia cell line . Expression can be modulated by injecting pro-myeloid factors like spi1b, which increases CCDC103 expression while decreasing gata1 expression, indicating a shift away from erythroid fate .

How do I analyze the effects of CCDC103 mutations on protein-protein interactions?

To analyze how CCDC103 mutations affect protein-protein interactions:

• Bioluminescence Resonance Energy Transfer (BRET) assay:

  • Clone wild-type and mutant CCDC103 into BRET donor vectors

  • Clone potential binding partners (e.g., DYNC1H1, SPAG6) into BRET acceptor vectors

  • Measure interaction strength through energy transfer efficiency

  • Research shows that PCD-causing mutations progressively reduce BRET signal intensity with both DYNC1H1 and SPAG6, with the most severe mutation (G128fs*) disrupting interactions completely

• Co-immunoprecipitation studies:

  • Express tagged versions of wild-type or mutant CCDC103

  • Immunoprecipitate using tag-specific antibodies

  • Analyze co-precipitated proteins by Western blotting

  • Include microtubule-stabilizing agents in lysis buffers to preserve interactions

• Proximity ligation assay (PLA):

  • Use antibodies against CCDC103 and potential binding partners

  • Perform in situ detection of protein-protein interactions in fixed cells

  • Quantify interaction signals and compare between wild-type and mutant conditions

• Microtubule co-sedimentation assay:

  • Polymerize purified tubulin in vitro

  • Add wild-type or mutant CCDC103 proteins

  • Sediment microtubules by ultracentrifugation

  • Analyze pellet and supernatant fractions for CCDC103 binding

Research has revealed that the severity of CCDC103 mutations correlates with the loss of interaction with both DYNC1H1 and SPAG6, with SPAG6 interactions being more sensitive to mutations than DYNC1H1 interactions .

What are the best model systems for studying CCDC103 function in myeloid cells?

Several model systems have proven valuable for investigating CCDC103 function in myeloid cells:

• Zebrafish models:

  • ccdc103/schmalhans (smh) mutants provide an established PCD model

  • Tg(spi1b:EGFP) transgenic lines allow visualization and isolation of myeloid lineages

  • Advantages include rapid development, transparent embryos for in vivo imaging, and ease of genetic manipulation

  • Studies in zebrafish have revealed that myeloid cells lacking CCDC103 show decreased proliferation, disrupted migration, and abnormal morphology

• Cell culture systems:

  • HL-60 cell line: can be differentiated into neutrophil-like or macrophage-like cells

  • Studies show CCDC103 localization differs between undifferentiated and differentiated HL-60 cells

  • Primary human CD34+ hematopoietic stem cells differentiated along myeloid lineages

• CRISPR/Cas9 engineered models:

  • Generate specific mutations matching human CCDC103 variants (H154P, G128fs*)

  • Create cell lines with fluorescent protein-tagged CCDC103 for live imaging

  • Develop conditional knockout systems to study temporal aspects of CCDC103 function

• Patient-derived systems:

  • Isolate myeloid cells from PCD patients with known CCDC103 mutations

  • Generate induced pluripotent stem cells (iPSCs) from patient samples and differentiate to myeloid lineages

Research using zebrafish spag6 mutants has shown that these fish recapitulate the functional defects in myeloid cells found in smh (CCDC103) mutants, providing valuable insight into the molecular mechanisms of CCDC103 function .

How can I distinguish between ciliary and non-ciliary functions of CCDC103?

Differentiating between ciliary and non-ciliary functions of CCDC103 requires specific experimental approaches:

• Cell type selection:

  • Study CCDC103 in non-ciliated cells (e.g., many lymphocytes, certain epithelial cells)

  • Compare functions in ciliated versus non-ciliated states of the same cell type

  • Investigate myeloid cells, which primarily exhibit non-ciliary CCDC103 functions

• Subcellular localization analysis:

  • Perform co-localization studies with ciliary markers (acetylated tubulin, IFT proteins)

  • Compare with cytoplasmic microtubule markers (α-tubulin) and dynein components

  • Research shows that in myeloid cells, CCDC103 localizes with cytoplasmic microtubules rather than primarily in cilia

• Domain-specific mutants:

  • Create constructs with mutations in domains specifically required for ciliary versus cytoplasmic functions

  • Perform rescue experiments in CCDC103-deficient models

  • Analyze specific functions (migration, proliferation, ciliary beating) separately

• Temporal manipulation:

  • Use inducible knockdown/knockout systems to deplete CCDC103 at different developmental stages

  • Analyze effects on established cilia versus cytoplasmic functions in mature cells

Research has established that CCDC103 has important cytoplasmic functions independent of its roles in motile cilia, including stabilizing microtubule-dynein interactions that regulate dynein-dependent processes within the cytoplasm, such as cargo transport and nuclear positioning .

How can CCDC103 antibodies be used to study myeloid cell migration defects?

CCDC103 antibodies can be powerful tools for investigating myeloid cell migration defects:

• Live-cell imaging approaches:

  • Use fluorescently labeled CCDC103 antibody fragments (Fab) for live imaging

  • Track co-localization with microtubules during cell migration

  • Correlate CCDC103 dynamics with migration velocity and directionality

  • Research shows CCDC103-deficient myeloid cells exhibit disrupted directed migration to sterile wound sites

• Fixed cell migration assays:

  • Perform wound healing or Transwell migration assays

  • Fix cells at different time points during migration

  • Immunostain for CCDC103 and cytoskeletal components

  • Quantify CCDC103 redistribution during polarization and migration

• In vivo migration tracking:

  • In zebrafish models, perform tail fin wounding assays

  • Track myeloid cell recruitment using lineage markers

  • Fix and immunostain for CCDC103 at various stages of the wound response

  • Compare wild-type and CCDC103 mutant cell behavior

• Correlation with microtubule dynamics:

  • Co-stain for CCDC103 and markers of dynamic microtubules (EB1, tyrosinated tubulin)

  • Assess microtubule stability using cold-resistant microtubule assays

  • Compare CCDC103 localization in migrating versus stationary cells

Studies in zebrafish ccdc103/smh mutants have demonstrated that myeloid cells lacking CCDC103 show disrupted directed migration to sterile wound sites and abnormal spherical morphology, consistent with loss of cytoplasmic microtubule stability .

What techniques can reveal CCDC103 interactions with the cytoskeleton?

Several advanced techniques can elucidate CCDC103 interactions with cytoskeletal components:

• Super-resolution microscopy:

  • Use structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM)

  • Visualize nanoscale co-localization of CCDC103 with microtubules

  • Track dynamic associations during cellular processes

  • Research shows CCDC103 puncta associate closely with the cytoplasmic microtubule network

• Biochemical fractionation:

  • Separate cytoskeletal and soluble fractions from cells

  • Analyze CCDC103 distribution by Western blotting

  • Compare distribution in normal cells versus after microtubule disruption with nocodazole

  • Research indicates nocodazole treatment disrupts CCDC103 interactions with binding partners

• Fluorescence recovery after photobleaching (FRAP):

  • Express fluorescently tagged CCDC103 in live cells

  • Photobleach regions associated with microtubules

  • Measure recovery kinetics to determine binding dynamics

  • Compare wild-type versus mutant CCDC103 mobility

• Microtubule co-sedimentation assays:

  • Purify recombinant CCDC103 (wild-type and mutants)

  • Mix with polymerized microtubules

  • Sediment complexes by ultracentrifugation

  • Analyze binding affinity and stoichiometry

Studies have shown that CCDC103 forms self-organizing oligomers and directly binds to and stabilizes cytoplasmic microtubules assembled in solution . In undifferentiated HL-60 myeloid progenitors, CCDC103 and cytoplasmic dynein heavy chain 1 (DYNC1H1) aggregates concentrate at putative microtubule-organizing centers (MTOCs) .

How do I troubleshoot inconsistent results when using CCDC103 antibodies?

When encountering inconsistent results with CCDC103 antibodies, consider these troubleshooting approaches:

• Antibody validation strategies:

  • Confirm specificity using CCDC103 knockout/knockdown controls

  • Test antibody on recombinant CCDC103 protein

  • Perform peptide competition assays to verify epitope specificity

  • Consider testing multiple antibodies targeting different epitopes

• Sample preparation optimization:

  • Test different lysis buffers that preserve microtubule integrity

  • Include protease inhibitors to prevent degradation

  • For immunofluorescence, compare different fixation methods (paraformaldehyde vs. methanol)

  • Consider cell-type specific differences in CCDC103 expression and localization

• Detection system adjustments:

  • For Western blotting, try different membrane types (PVDF vs. nitrocellulose)

  • Optimize blocking conditions (milk vs. BSA)

  • Test various detection systems (chemiluminescence, fluorescence)

  • For immunofluorescence, compare different mounting media to preserve signal

• Biological variability considerations:

  • CCDC103 localization differs between cell types and differentiation states

  • Expression levels vary across developmental stages and cell lineages

  • Microtubule association may be dynamic and affected by cell cycle stage

Research shows that CCDC103 exhibits different localization patterns in undifferentiated versus differentiated myeloid cells. In undifferentiated HL-60 myeloid progenitor cells, CCDC103 localizes in larger, more sparse puncta, whereas in differentiated cells, it shows smaller punctate and more diffuse distribution .

How can CCDC103 antibodies help investigate Primary Ciliary Dyskinesia (PCD)?

CCDC103 antibodies offer valuable tools for PCD research:

• Diagnostic applications:

  • Immunostaining of patient ciliated epithelial cells

  • Western blot analysis of CCDC103 protein levels in patient samples

  • Correlation of protein expression/localization with specific CCDC103 mutations

  • Research shows mutations in CCDC103 cause highly variable clinical presentations in PCD patients

• Functional assessments:

  • Compare CCDC103 localization in normal versus PCD patient samples

  • Correlate CCDC103 antibody staining patterns with ciliary ultrastructure

  • Assess protein-protein interactions in patient-derived materials

  • Studies show patient mutations in CCDC103 disrupt microtubule-dependent interactions with both DYNC1H1 and SPAG6

• Model system validation:

  • Verify CCDC103 knockout/knockdown/mutation in animal and cell models

  • Compare antibody staining patterns between patient samples and model systems

  • Evaluate rescue experiments with wild-type CCDC103 expression

• Therapeutic development support:

  • Monitor restoration of CCDC103 expression in gene therapy approaches

  • Evaluate protein-protein interactions after therapeutic interventions

  • Assess normalization of subcellular localization

Research has identified three known alleles in CCDC103 that cause PCD in humans, with varying clinical presentations. The severity of mutations correlates with the loss of interaction between CCDC103 and its binding partners DYNC1H1 and SPAG6 .

What is the role of CCDC103 in sperm flagella and how can antibodies aid fertility research?

CCDC103 plays a crucial role in sperm flagella, and antibodies can advance fertility research:

• Comparative ultrastructural analysis:

  • Immunostain sperm samples for CCDC103 localization

  • Correlate with flagellar ultrastructure by electron microscopy

  • Compare normal sperm with samples from infertile patients

  • Research shows the CCDC103 p.His154Pro variant affects both cilia and sperm flagellum

• Mutation-specific effects:

  • Analyze CCDC103 in sperm from patients with specific mutations

  • Compare antibody staining patterns across different mutation types

  • Correlate with sperm motility parameters

  • Studies report total sperm immotility associated with the CCDC103 p.His154Pro mutation in men with normal respiratory phenotypes

• Differential diagnosis approaches:

  • Use CCDC103 antibodies to distinguish between different causes of immotile sperm

  • Compare with other dynein assembly factor defects

  • Correlate antibody staining with specific ultrastructural abnormalities

  • Research reveals different degrees of ultrastructural abnormalities in sperm with CCDC103 mutations

• Therapeutic target validation:

  • Assess CCDC103 in experimental fertility treatments

  • Monitor protein expression and localization after interventions

  • Correlate with functional recovery of sperm motility

The CCDC103 p.His154Pro variant has been shown to affect both cilia and sperm flagellum, suggesting this mutation acts in a shared pathway of dynein arms formation in both cell types . Research has documented total sperm immotility associated with this mutation, even in men with normal respiratory phenotypes, indicating tissue-specific effects of certain CCDC103 mutations .

How can I correlate CCDC103 expression with myeloid cell dysfunction in disease models?

To correlate CCDC103 expression with myeloid dysfunction:

• Patient-derived myeloid cell analysis:

  • Isolate myeloid cells from PCD patients with CCDC103 mutations

  • Assess migration, proliferation, and morphology compared to healthy controls

  • Quantify CCDC103 expression/localization by immunostaining and Western blotting

  • Research shows myeloid cells lacking CCDC103 have decreased proliferation and disrupted migration

• Cytokine response evaluation:

  • Stimulate myeloid cells with inflammatory cytokines

  • Monitor CCDC103 expression and localization changes

  • Correlate with functional responses (phagocytosis, cytokine production)

  • Compare wild-type versus CCDC103-deficient cells

• In vivo myeloid tracking:

  • In zebrafish or mouse models with CCDC103 mutations

  • Track myeloid cell recruitment to infection or injury sites

  • Fixed-tissue immunostaining to correlate CCDC103 with myeloid dysfunction

  • Studies in zebrafish found myeloid cells lacking CCDC103 show disrupted directed migration to sterile wound sites

• Bone marrow transplantation models:

  • Transplant CCDC103-deficient bone marrow into wild-type recipients

  • Analyze reconstituted myeloid cells for migration and function

  • Use CCDC103 antibodies to confirm persistent deficiency

  • Evaluate tissue-specific myeloid dysfunction

Research has established that CCDC103 has important roles in myeloid cell function independent of its ciliary functions. Zebrafish ccdc103/smh mutants display decreased myeloid cell proliferation, disrupted migration, and abnormal morphology consistent with cytoplasmic microtubule instability .

How might CCDC103 research contribute to understanding broader cytoskeletal regulation?

CCDC103 research offers insights into cytoskeletal regulation:

• Microtubule stabilization mechanisms:

  • Investigate how CCDC103 oligomers stabilize microtubules

  • Compare with other microtubule-associated proteins (MAPs)

  • Examine potential cooperative effects with SPAG6

  • Research shows CCDC103 forms self-organizing oligomers and directly binds to and stabilizes cytoplasmic microtubules

• Dynein regulation pathways:

  • Explore how CCDC103 affects dynein motor activity

  • Investigate connections between ciliary and cytoplasmic dynein regulation

  • Examine effects on cargo transport in non-ciliated cells

  • Studies indicate CCDC103 may stabilize microtubule-dynein interactions that regulate dynein-dependent cellular processes

• Cell-type specific cytoskeletal requirements:

  • Compare CCDC103 function across diverse cell types

  • Identify tissue-specific binding partners and effects

  • Relate to specialized cytoskeletal arrangements in different cells

  • Research reveals different CCDC103 localization patterns and functions across cell types

• Evolutionary conservation analysis:

  • Examine CCDC103 orthologs across species

  • Correlate structural features with functional conservation

  • Identify domains selectively maintained in evolution

CCDC103 research suggests it may serve as a scaffolding or adaptor protein that facilitates the function of multiple proteins requiring microtubule localization, with implications for understanding fundamental cytoskeletal organization principles .

What emerging technologies might enhance CCDC103 antibody-based research?

Emerging technologies promising for CCDC103 antibody research include:

• Single-molecule imaging techniques:

  • Apply super-resolution microscopy to visualize individual CCDC103 molecules

  • Track dynamic associations with microtubules and motor proteins

  • Correlate molecular behavior with cellular functions

  • Could reveal detailed mechanisms of how CCDC103 stabilizes microtubules

• Mass spectrometry immunoprecipitation (MS-IP):

  • Use CCDC103 antibodies to isolate native protein complexes

  • Identify novel binding partners through unbiased proteomic analysis

  • Compare interactomes across different cell types and conditions

  • May discover additional interactions beyond known partners DYNC1H1 and SPAG6

• Intrabody applications:

  • Develop cell-permeable CCDC103 antibody fragments

  • Use for live imaging and functional perturbation

  • Target specific domains to disrupt select protein interactions

  • Could distinguish between different functional domains of CCDC103

• Cryo-electron tomography:

  • Visualize CCDC103-microtubule complexes at near-atomic resolution

  • Determine structural basis for microtubule stabilization

  • Compare wild-type and mutant protein structures

  • May reveal how mutations disrupt critical protein-protein interactions

These technologies could provide unprecedented insights into CCDC103's roles in both ciliary and non-ciliary contexts, potentially revealing new therapeutic targets for PCD and related disorders.

How might comparative studies of CCDC103 and SPAG6 advance our understanding of microtubule regulation?

Comparative studies of CCDC103 and SPAG6 offer promising research directions:

• Cooperative binding analysis:

  • Investigate whether CCDC103 and SPAG6 bind microtubules cooperatively

  • Determine if they recognize distinct or overlapping binding sites

  • Assess combined effects on microtubule stability

  • Research shows SPAG6 is a novel CCDC103-binding partner that promotes microtubule stability

• Mutation impact comparison:

  • Compare effects of CCDC103 and SPAG6 mutations on shared cellular processes

  • Assess whether mutations affecting their interaction cause similar phenotypes

  • Research shows engineered zebrafish spag6 mutants recapitulate functional defects in myeloid cells found in CCDC103-deficient smh mutants

• Tissue-specific expression patterns:

  • Map co-expression of CCDC103 and SPAG6 across tissues and developmental stages

  • Identify contexts where their functions may be redundant versus complementary

  • Correlate expression patterns with microtubule-dependent cell functions

• Molecular rescue experiments:

  • Test whether SPAG6 overexpression can rescue CCDC103 deficiency phenotypes

  • Investigate domain-specific requirements for functional complementation

  • Design chimeric proteins to determine functional domain equivalence