CFAP61 Antibody

What is CFAP61 and what cellular functions does it regulate?

CFAP61 (Cilia and Flagella Associated Protein 61) is a protein that plays critical roles in cell motility and ciliary function. It specifically regulates ciliary beating and morphogenesis, making it essential for proper mucociliary clearance in respiratory tissues and flagellar function in reproductive cells. The protein was previously designated as an uncharacterized protein C20orf26 homolog before its functional characterization .

Research with knockout models has demonstrated that CFAP61 is particularly important for sperm flagella development and function, with deficiencies leading to multiple morphological abnormalities of sperm flagella (MMAF) . The protein's involvement in ciliary architecture maintenance suggests it contributes to both structural integrity and functional dynamics of motile cilia.

What types of CFAP61 antibodies are available for research applications?

Several types of CFAP61 antibodies are available for research applications, with polyclonal rabbit antibodies being particularly common. These include:

• Polyclonal antibodies raised in rabbits (e.g., CAB18194) with specific reactivity for human CFAP61

• Antibodies validated for various applications including Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA)

• Antibodies with different species reactivity profiles (human-specific, mouse-specific, or cross-reactive with multiple species)

The selection of an appropriate antibody depends on the specific research application, desired species reactivity, and validation requirements for the study design.

How is CFAP61 protein structurally characterized and what are its key domains?

While the search results don't provide detailed structural information about CFAP61's domains, we know it functions in ciliary and flagellar contexts. Proteins in this family typically contain structural motifs that facilitate interactions with microtubules, other axonemal proteins, or regulatory molecules involved in ciliary beating.

Functional studies suggest CFAP61 likely contains domains involved in:

• Microtubule binding or stabilization, particularly affecting the central pair microtubules of the axoneme

• Protein-protein interaction regions that facilitate assembly of the ciliary/flagellar apparatus

• Potential regulatory domains that coordinate ciliary beating patterns

Further structural characterization through techniques like X-ray crystallography or cryo-electron microscopy would be valuable for elucidating the precise domain architecture of CFAP61.

What experimental protocols yield optimal results when using CFAP61 antibodies for immunofluorescence?

For optimal immunofluorescence results with CFAP61 antibodies, researchers should consider the following methodological approaches:

• Sample Preparation:

  • For sperm samples: Fix with 4% paraformaldehyde, followed by permeabilization with 0.1-0.5% Triton X-100

  • For respiratory epithelial cells: Gentle fixation protocols are recommended to preserve ciliary structures

• Antibody Concentration and Incubation:

  • Typical dilutions range from 1:100 to 1:500 for primary CFAP61 antibodies

  • Overnight incubation at 4°C often yields superior signal-to-noise ratios

  • Include appropriate blocking steps with BSA or normal serum to minimize non-specific binding

• Counterstaining Strategies:

  • Combine with acetylated tubulin staining to visualize the entire ciliary/flagellar structure

  • DAPI nuclear counterstaining provides cellular context

  • In sperm studies, mitochondrial markers can help assess the integrity of the mitochondrial sheath

The immunofluorescence pattern of CFAP61 typically shows localization along the axoneme of cilia and flagella, with reduced or absent staining observed in samples from individuals with pathogenic CFAP61 variants .

What are the recommended Western blot conditions for detecting CFAP61 protein?

For optimal Western blot detection of CFAP61 protein:

• Sample Preparation:

  • For ciliated tissue or cells: Direct lysis in RIPA buffer supplemented with protease inhibitors

  • For sperm samples: Special extraction buffers may be required to solubilize axonemal proteins

• Electrophoresis Parameters:

  • Use 8-10% SDS-PAGE gels due to the relatively large size of CFAP61

  • Longer running times may improve resolution

• Transfer and Detection:

  • Semi-dry or wet transfer systems with PVDF membranes are suitable

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Incubation overnight at 4°C often improves specific binding

  • HRP-conjugated secondary antibodies with ECL detection provide good sensitivity

• Controls and Validation:

  • Include positive controls from tissues known to express CFAP61 (testis, respiratory epithelium)

  • Negative controls should include samples from CFAP61-knockout models or tissues known to lack expression

  • Expect a band corresponding to the predicted molecular weight of CFAP61

These conditions may need optimization based on the specific CFAP61 antibody being used and the particular sample type under investigation .

How can researchers verify the specificity of CFAP61 antibodies in their experimental system?

Verifying antibody specificity is crucial for reliable CFAP61 research. Recommended validation approaches include:

• Genetic Controls:

  • Use tissue/cells from CFAP61 knockout or knockdown models as negative controls

  • Compare staining patterns in samples with known CFAP61 variants versus wild-type samples

• Multiple Antibody Verification:

  • Utilize antibodies targeting different epitopes of CFAP61 to confirm consistent localization patterns

  • Compare results from different antibody types (monoclonal vs. polyclonal)

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess purified CFAP61 protein or peptide

  • Specific antibodies will show reduced or eliminated signal after competition

• Correlation with mRNA Expression:

  • Verify that antibody signal correlates with CFAP61 mRNA expression patterns across tissues

• Immunoprecipitation Followed by Mass Spectrometry:

  • Confirm that the immunoprecipitated protein is indeed CFAP61 through peptide mass fingerprinting

These validation steps ensure that experimental observations genuinely reflect CFAP61 biology rather than antibody cross-reactivity or non-specific binding .

How do pathogenic variants in CFAP61 contribute to male infertility phenotypes?

Pathogenic variants in CFAP61 have been directly linked to male infertility through the following mechanisms:

• Sperm Flagellar Structural Abnormalities:

  • Bi-allelic CFAP61 variants cause multiple morphological abnormalities of the sperm flagella (MMAF)

  • Electron microscopy reveals absence of central pair microtubules in the axoneme

  • Mitochondrial sheath malformation occurs in the flagella

• Functional Impact on Sperm:

  • Affected individuals present with oligoasthenoteratozoospermia (OAT), characterized by:

    • Low sperm count (oligozoospermia)

    • Poor motility (asthenozoospermia)

    • Abnormal morphology (teratozoospermia)

• Genetic Evidence:

  • Specific variants identified include: c.1654C>T (p.R552C), c.2911G>A (p.D971N), c.144-2A>G, and c.1666G>A (p.G556R)

  • In silico and in vitro functional analyses confirmed the deleterious effects of these variants

  • The infertility phenotype is recessive, requiring bi-allelic variants

The direct causative relationship between CFAP61 dysfunction and male infertility has been further supported by animal models, as Cfap61-deficient mice also display the OAT phenotype .

Can CFAP61 antibodies be used in diagnostic applications for ciliopathies?

While not yet established as standard clinical diagnostics, CFAP61 antibodies hold potential for diagnostic applications in ciliopathies through several approaches:

• Immunohistochemical Assessment:

  • CFAP61 antibodies can detect altered expression or localization in ciliated tissues from patients with suspected ciliopathies

  • Reduced or absent CFAP61 staining in otherwise intact cilia could indicate pathogenic variants

• Sperm Analysis in Male Infertility:

  • Immunofluorescence assays with CFAP61 antibodies reveal "markedly reduced CFAP61 staining in the sperm flagella" of affected individuals

  • This could complement genetic testing for male infertility diagnosis

• Research Applications Informing Future Diagnostics:

  • Characterizing the relationship between specific CFAP61 variants and protein expression/localization patterns

  • Developing antibody panels that target multiple ciliary proteins including CFAP61

Before clinical implementation, extensive validation of antibody performance across diverse patient samples would be required. Integration with genetic testing would likely provide the most comprehensive diagnostic approach for CFAP61-related ciliopathies .

What is the relationship between CFAP61 dysfunction and respiratory disease pathogenesis?

The relationship between CFAP61 dysfunction and respiratory disease stems from its critical role in ciliary function:

Future research using CFAP61 antibodies to examine respiratory epithelial samples from patients with unexplained respiratory conditions could help elucidate the full spectrum of CFAP61-related respiratory pathologies .

What approaches can be used to study CFAP61 protein interactions within the ciliary axoneme complex?

Advanced approaches to study CFAP61 protein interactions within the ciliary axoneme include:

• Proximity Labeling Techniques:

  • BioID or APEX2 fusion proteins with CFAP61 to identify proximal interacting partners in living cells

  • TurboID offers faster labeling kinetics for capturing transient interactions

• Co-immunoprecipitation with CFAP61 Antibodies:

  • Gentle lysis conditions to preserve native protein complexes

  • Mass spectrometry identification of co-precipitated proteins

  • Reciprocal co-IP validation of key interactions

• Super-resolution Microscopy:

  • STORM or PALM imaging using CFAP61 antibodies combined with other axonemal protein markers

  • Provides spatial resolution to map CFAP61 positioning relative to other axonemal components

• Cryo-electron Tomography:

  • Structural analysis of wild-type versus CFAP61-deficient axonemes

  • Can reveal the precise location of CFAP61 within the 3D axonemal architecture

• Functional Interaction Studies:

  • Mutagenesis of potential interaction domains followed by phenotypic assessment

  • FRET-based approaches to study direct interactions with candidate partners

These approaches can collectively build a comprehensive interactome map for CFAP61, revealing its functional integration within the complex ciliary machinery .

How can researchers integrate CFAP61 antibody studies with genetic analysis in ciliopathy research?

Integrating CFAP61 antibody studies with genetic analysis creates a powerful approach for ciliopathy research:

• Genotype-Phenotype Correlation Studies:

  • Use CFAP61 antibodies to assess protein expression/localization in samples with different CFAP61 variants

  • Create a catalog linking specific variants to particular patterns of protein dysfunction

  • Example: Specific variants (c.1654C>T, c.2911G>A, c.144-2A>G, c.1666G>A) have been linked to male infertility with demonstrable changes in CFAP61 localization

• Functional Validation of Variants of Uncertain Significance (VUS):

  • Apply CFAP61 antibodies to assess the impact of novel variants on protein expression and localization

  • Combine with in vitro functional assays to determine pathogenicity

• CRISPR-engineered Cell/Animal Models:

  • Generate models with specific CFAP61 variants identified in patients

  • Use antibodies to compare protein dynamics between wild-type and mutant models

  • Assess rescue with wild-type CFAP61 expression

• Multi-omic Integration:

  • Correlate CFAP61 antibody staining patterns with transcriptomic and proteomic changes

  • Identify compensatory mechanisms in response to CFAP61 dysfunction

• Family-based Studies:

  • Analyze CFAP61 expression/localization in samples from affected and unaffected family members

  • Correlate with inheritance patterns of specific variants

This integrated approach provides more comprehensive insights than either genetic or antibody-based methods alone .

What experimental considerations are important when studying species-specific differences in CFAP61 function using antibodies?

When investigating species-specific differences in CFAP61 function using antibodies, researchers should consider:

• Antibody Cross-reactivity Assessment:

  • Verify whether human-targeted CFAP61 antibodies recognize mouse or other species' orthologs

  • Consider using species-specific antibodies when available (human-specific, mouse-specific)

  • Perform Western blot validation across species before immunofluorescence applications

• Sequence Homology Analysis:

  • Assess conservation of the epitope region across species

  • Higher sequence divergence may necessitate species-specific antibodies

  • Consider that CFAP61 was previously designated as "uncharacterized protein C20orf26 homolog" in non-human species

• Functional Conservation Evaluation:

  • Compare localization patterns across species using species-appropriate antibodies

  • Assess whether phenotypes of CFAP61 dysfunction are consistent across species

  • Mouse models show similar infertility phenotypes to humans with CFAP61 variants, suggesting functional conservation

• Tissue-specific Expression Patterns:

  • Investigate whether CFAP61 expression patterns in ciliated tissues are consistent across species

  • Document any species-specific differences in subcellular localization

• Control Selection:

  • Include appropriate positive and negative controls for each species

  • Consider using CFAP61-knockout tissues from the relevant species as definitive negative controls

These considerations ensure that observed differences reflect genuine biological variation rather than technical limitations of the antibodies used .