CFAP65 Antibody

What is CFAP65 and why is it significant in research?

CFAP65 (cilia and flagella associated protein 65) is a fundamental protein involved in the development and formation of ciliated flagella. In humans, the canonical protein has 1925 amino acid residues with a mass of 217.3 kDa and is primarily localized in cell projections and cytoplasm. It plays a critical role in flagellar formation, sperm motility, and basal body migration/docking to the plasma membrane. CFAP65 is also involved in apical enrichment of F-actin during multiciliogenesis. Recent research has revealed its potential significance in cancer biology, particularly as a prognostic marker in colon cancer .

How many isoforms of CFAP65 exist and what are their functional differences?

Up to four different isoforms of CFAP65 have been reported in humans. The functional differences between these isoforms relate to their specific roles in various cellular processes, including cell motility, ion channel function, and GTPase-associated signaling. When designing experiments with CFAP65 antibodies, researchers should consider which isoform(s) they need to target based on tissue-specific expression patterns and functional relevance to their research question .

What are the most suitable applications for CFAP65 antibodies in basic research?

CFAP65 antibodies are most commonly used in Western Blot, ELISA, and Immunohistochemistry applications. For basic research characterizing CFAP65 expression and localization, Western Blot provides quantitative protein expression data, while immunohistochemistry and immunofluorescence allow visualization of subcellular localization in cell projections and cytoplasm. ELISA applications are valuable for quantitative detection in solution-based samples. The choice of application should align with your specific research objectives and sample types .

How should I optimize Western Blot protocols for CFAP65 detection?

For optimal Western Blot detection of CFAP65:

• Sample preparation: Use RIPA buffer with protease inhibitors for tissue/cell lysis

• Gel selection: Due to the large size of CFAP65 (217.3 kDa), use low percentage (6-8%) SDS-PAGE gels

• Transfer conditions: Employ extended transfer times (overnight at low voltage) or semi-dry transfer systems optimized for high molecular weight proteins

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute according to manufacturer's recommendation (typically 1:500-1:2000) and incubate overnight at 4°C

• Visualization: Use secondary antibodies appropriate for your detection system and primary antibody host species

Validation controls should include positive control tissues known to express CFAP65 (such as testicular tissue) and negative controls where the primary antibody is omitted .

What considerations are important when using CFAP65 antibodies for immunohistochemistry?

When performing immunohistochemistry with CFAP65 antibodies:

• Fixation: 10% neutral buffered formalin (24-48 hours) is recommended

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking: Use species-appropriate serum (5-10%) to reduce background

• Antibody dilution: Typically 1:100-1:500 based on antibody specifications

• Incubation: Overnight at 4°C for optimal binding

• Detection system: Choose chromogenic (DAB) or fluorescent detection based on research needs

• Counterstaining: Hematoxylin for chromogenic or DAPI for fluorescent detection

For scoring CFAP65 expression in IHC samples, consider both staining intensity (0-3+) and percentage of positive cells to develop a composite score. This approach has been effective in studies evaluating CFAP65 as a prognostic marker in colon cancer .

How does conjugate selection affect experimental outcomes when using CFAP65 antibodies?

Conjugate selection significantly impacts experimental outcomes with CFAP65 antibodies:

• Unconjugated antibodies: Most versatile, allowing researcher-selected secondary detection systems, but require additional incubation steps

• HRP-conjugated antibodies: Ideal for ELISA and chromogenic IHC/WB applications, with direct enzymatic detection

• Biotin-conjugated antibodies: Allow signal amplification through streptavidin systems, enhancing sensitivity

• Fluorophore-conjugated antibodies: Enable direct visualization in IF/ICC applications and flow cytometry

The optimal conjugate depends on your specific application, detection system, and whether multiplexing is required. For co-localization studies examining CFAP65 interactions with other ciliary proteins, fluorophore-conjugated antibodies allow for simultaneous detection of multiple targets .

How can I effectively design experiments to study CFAP65's role in ciliopathies?

To effectively study CFAP65's role in ciliopathies:

• Model selection:

  • Patient-derived samples (if available)

  • CRISPR-Cas9 CFAP65 knockout/knockdown cell lines

  • Animal models (zebrafish, mouse) with CFAP65 mutations

• Experimental approaches:

  • Phenotypic characterization: Assess cilia formation, length, and motility

  • Live-cell imaging with fluorescently tagged CFAP65

  • Co-immunoprecipitation to identify CFAP65 binding partners

  • Proximity ligation assays to confirm protein-protein interactions in situ

• Functional readouts:

  • Cilia beat frequency analysis

  • Intraflagellar transport (IFT) tracking

  • Developmental phenotypes in animal models

  • Cell migration and polarization assays

• Data analysis:

  • Quantitative assessment of cilia parameters (length, number, morphology)

  • Statistical comparison between wild-type and mutant models

  • Correlation of molecular findings with phenotypic outcomes

This comprehensive approach enables researchers to connect molecular mechanisms to disease manifestations in ciliopathies associated with CFAP65 dysfunction .

How should researchers interpret contradictory CFAP65 expression data between different cancer types?

When confronted with contradictory CFAP65 expression data across cancer types:

• Contextualize findings within tissue-specific biology:

  • Consider the normal role of cilia in the tissue of origin

  • Evaluate tissue-specific CFAP65 isoform expression profiles

  • Assess correlation with other ciliary genes in the dataset

• Methodological reconciliation:

  • Compare antibody clones and detection methods used

  • Evaluate sample preparation and antigen retrieval protocols

  • Review scoring systems and thresholds for "high" vs. "low" expression

• Biological interpretation framework:

  • Primary cilia may have tumor-promoting or tumor-suppressive functions depending on context

  • CFAP65 could participate in non-ciliary functions through GTPase-associated signaling

  • Expression changes may represent consequences rather than drivers of tumorigenesis

• Validation approaches:

  • Perform parallel analysis using multiple detection methods (IHC, WB, qPCR)

  • Correlate protein expression with RNA-seq data

  • Functional studies to determine the effect of CFAP65 modulation on cancer phenotypes

The apparent contradictions may reveal important tissue-specific functional differences. For example, CFAP65's association with better prognosis in colon cancer should be interpreted within the context of colon-specific ciliary functions and signaling pathways .

What advanced techniques can be used to study CFAP65's protein-protein interactions in cilia assembly?

Advanced techniques for studying CFAP65's protein-protein interactions include:

• Proximity-based interaction mapping:

  • BioID or TurboID fusion proteins to identify proximal interactors

  • APEX2-based proximity labeling coupled with mass spectrometry

  • FRET/FLIM analysis for direct protein-protein interactions in live cells

• Pull-down approaches:

  • Tandem affinity purification with epitope-tagged CFAP65

  • Co-immunoprecipitation with endogenous CFAP65 antibodies

  • GST-fusion protein interaction assays for domain-specific interactions

• Structural analysis:

  • Cryo-EM of purified CFAP65-containing complexes

  • Cross-linking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding-induced conformational changes

• Live-cell dynamics:

  • Fluorescence recovery after photobleaching (FRAP) to measure CFAP65 dynamics

  • Single-molecule tracking of tagged CFAP65 during ciliogenesis

  • Optogenetic approaches to temporally control CFAP65 interactions

These techniques provide complementary data on the spatiotemporal dynamics of CFAP65 interactions during cilia assembly and function, helping to elucidate its role in basal body migration and docking to the plasma membrane .

How does CFAP65 expression correlate with clinical outcomes in colon cancer?

CFAP65 expression shows significant correlation with clinical outcomes in colon cancer:

CFAP65 expression was significantly higher in colon cancer tissue compared to matched paratumor tissue. In clinical samples, 61.9% showed high expression while 38.1% showed low expression. Cox hazard regression analysis identified CFAP65 expression as an independent prognostic factor along with tumor stage and tumor location.

What methodological approaches can determine if CFAP65 has a functional role in cancer progression versus serving as a biomarker?

To distinguish between CFAP65's potential functional role in cancer versus its utility as a biomarker:

• Functional validation studies:

  • CRISPR-Cas9 knockout/knockdown in cancer cell lines followed by:

    • Proliferation, migration, and invasion assays

    • Tumor formation in xenograft models

    • Drug sensitivity testing

  • Overexpression studies in low-expressing cell lines

  • Rescue experiments with wild-type vs. mutant CFAP65

• Mechanistic investigations:

  • Analysis of downstream signaling pathways (particularly GTPase-associated)

  • Assessment of cell motility and cytoskeletal organization

  • Evaluation of changes in primary cilia structure and signaling

  • Identification of cancer-specific CFAP65 binding partners

• Temporal expression analysis:

  • CFAP65 expression at different stages of tumor progression

  • Expression changes in response to therapy

  • Correlation with tumor evolution in longitudinal samples

• Clinical correlation:

  • Multivariate analysis controlling for known prognostic factors

  • Integration with molecular subtyping data

  • Correlation with treatment response metrics

The finding that CFAP65-related genes are significantly enriched in cellular processes of cell motility, ion channels, and GTPase-associated signaling provides mechanistic avenues to explore for functional studies .

How should researchers design experiments to investigate CFAP65's potential as a therapeutic target?

To investigate CFAP65's potential as a therapeutic target, researchers should design experiments following this framework:

• Target validation:

  • Determine tissue/tumor specificity of CFAP65 expression

  • Assess essentiality through CRISPR screens in multiple cell lines

  • Evaluate potential on-target toxicity in normal tissues

  • Conduct synthetic lethality screens to identify context-dependent vulnerabilities

• Therapeutic strategy development:

  • Domain-specific inhibition (identify druggable domains)

  • Protein-protein interaction disruption

  • Degradation approaches (PROTACs, molecular glues)

  • Antibody-drug conjugates if cell-surface expression is confirmed

• Preclinical efficacy studies:

  • Develop appropriate in vitro and in vivo models

  • Test combination approaches with standard-of-care therapies

  • Evaluate resistance mechanisms

  • Identify biomarkers of response

• Translational considerations:

  • Develop companion diagnostics for CFAP65 expression/activity

  • Establish clinically relevant cutoffs for "high" vs. "low" expression

  • Design appropriate patient selection strategies

  • Plan for pharmacodynamic markers in early clinical trials

The design should account for CFAP65's roles in both normal ciliary function and cancer-specific contexts to maximize therapeutic window and minimize potential adverse effects .

What are the most common technical challenges in CFAP65 detection and how can they be addressed?

Common technical challenges in CFAP65 detection and their solutions include:

• High molecular weight detection issues:

  • Challenge: Poor transfer of 217.3 kDa protein in Western blots

  • Solutions:

    • Use gradient gels (4-12%)

    • Extend transfer time (overnight at 30V)

    • Employ specialized transfer buffers with reduced methanol

    • Consider wet transfer systems over semi-dry for large proteins

• Antibody specificity concerns:

  • Challenge: Cross-reactivity with other CFAP family members

  • Solutions:

    • Validate antibodies using CFAP65 knockout/knockdown controls

    • Confirm detection of expected size band in Western blot

    • Compare results from multiple antibody clones

    • Pre-absorb antibodies with recombinant related proteins

• Low signal strength:

  • Challenge: Weak detection in tissues with moderate expression

  • Solutions:

    • Optimize antigen retrieval (test multiple buffers and pH)

    • Increase antibody concentration and incubation time

    • Use signal amplification systems (TSA, ABC method)

    • Fresh sample collection and proper fixation protocols

• Background issues in IHC/IF:

  • Challenge: Non-specific staining obscuring true signal

  • Solutions:

    • Extend blocking time (2-3 hours)

    • Use multiple blocking agents (normal serum + BSA)

    • Include detergents in wash buffers

    • Optimize secondary antibody dilutions

These optimizations are particularly important when studying CFAP65 in contexts where expression levels may vary significantly, such as in comparative studies between tumor and normal tissues .

How can researchers validate the specificity of their CFAP65 antibody across different experimental conditions?

Comprehensive CFAP65 antibody validation across experimental conditions requires:

• Expression system controls:

  • Positive controls: Tissues/cells with known high CFAP65 expression (testis, ciliated epithelia)

  • Negative controls: CFAP65 knockout/knockdown models

  • Overexpression controls: Cells transfected with CFAP65 expression constructs

  • Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

• Cross-platform validation:

  • Correlation between protein detection (IHC/WB) and mRNA expression (qPCR/RNA-seq)

  • Consistency across multiple detection methods (IF vs. IHC vs. WB)

  • Comparison of staining patterns between monoclonal and polyclonal antibodies

  • Verification with alternative antibody clones targeting different epitopes

• Protocol-specific validation:

  • Test antibody performance across different fixation methods

  • Verify specificity under varied antigen retrieval conditions

  • Assess performance in fresh vs. archived samples

  • Determine optimal antibody concentration for each application

• Specificity documentation:

  • Record exact conditions where specificity was confirmed

  • Document lot-to-lot variability

  • Maintain validation data with experimental samples

  • Include validation controls in publications

This rigorous validation approach ensures reliable detection of CFAP65 across experimental systems and applications, critical for reproducible research outcomes .

What methodological considerations are important when studying CFAP65 post-translational modifications?

When investigating CFAP65 post-translational modifications (PTMs), researchers should consider:

• Sample preparation:

  • Use phosphatase inhibitors for phosphorylation studies

  • Include deubiquitinase inhibitors for ubiquitination analysis

  • Consider native conditions to preserve protein complexes

  • Employ specific lysis buffers based on the targeted PTM

• Enrichment strategies:

  • Phospho-enrichment: IMAC or titanium dioxide chromatography

  • Ubiquitinated protein enrichment: TUBE technology or specific antibodies

  • SUMOylated protein isolation: Ni-NTA purification of His-tagged SUMO

  • Immunoprecipitation with PTM-specific antibodies prior to CFAP65 detection

• Detection approaches:

  • Western blot with phospho-specific antibodies

  • Mass spectrometry for unbiased PTM mapping

  • Proximity ligation assays for in situ PTM detection

  • 2D gel electrophoresis for charge-based separation of modified forms

• Functional validation:

  • Site-directed mutagenesis of modified residues

  • Pharmacological inhibition of modifying enzymes

  • Temporal analysis during ciliogenesis

  • Correlation of modifications with CFAP65 activity or localization

Understanding CFAP65 PTMs may provide insight into its regulation during ciliogenesis and its potential dysregulation in disease states. The large size and multiple domains of CFAP65 suggest it likely undergoes complex post-translational regulation that could significantly impact its function in both normal and pathological conditions .