CFAP52 Antibody

What is CFAP52 and what cellular functions does it perform?

CFAP52 (cilia and flagella associated protein 52) is a WD repeat-containing protein that functions as a microtubule inner protein (MIP) within the dynein-decorated doublet microtubules (DMTs) in cilia axoneme. The protein is required for proper motile cilia beating and plays critical roles in ciliary/flagellar structure and function . In humans, the canonical CFAP52 protein has 620 amino acid residues with a molecular mass of 68.3 kDa and contains WD repeat domains that serve as protein interaction scaffolds in multiprotein complexes . It primarily localizes to the cytoplasm and is predominantly expressed in respiratory cells and sperm .

What are the common applications for CFAP52 antibodies in research?

CFAP52 antibodies are primarily used in several key research applications:

• Western Blotting: For detecting CFAP52 protein expression in tissue lysates, particularly in testis and respiratory tissues

• Immunofluorescence: For visualizing the subcellular localization of CFAP52 in sperm cells and ciliated tissues

• Immunohistochemistry: For examining CFAP52 expression patterns in tissue sections

These applications are essential for studying CFAP52's role in sperm development, cilia formation, and related pathologies .

What species reactivity is available for CFAP52 antibodies?

Based on the available information, CFAP52 antibodies exhibit reactivity across multiple species:

Most comprehensive antibodies show reactivity to human, mouse, and rat CFAP52, with some antibodies demonstrating broader cross-reactivity . When selecting an antibody for your research, it's important to verify the specific species reactivity in the product documentation.

How do I optimize immunofluorescence staining protocols for CFAP52 detection in sperm samples?

For optimal immunofluorescence staining of CFAP52 in sperm samples:

• Prepare sperm slides and permeabilize the cell membranes.

• Block with 5% goat serum for approximately 45 minutes to reduce non-specific binding .

• Incubate with primary CFAP52 antibody overnight at 4°C (dilutions typically range from 1:100 to 1:500, but optimize based on your specific antibody).

• Wash thoroughly with PBS (3-5 times) to remove unbound antibody.

• Incubate with fluorophore-conjugated secondary antibodies (Alexa Fluor 484 or 555) for 45 minutes at room temperature .

• Counterstain nuclei with DAPI.

• Mount slides with anti-fade mounting medium.

For sperm samples specifically, CFAP52 signals should be visible at both the head-tail connecting apparatus (HTCA) and along the full-length flagella in normal sperm, serving as an important control comparison when studying samples with potential CFAP52 deficiencies .

What is the role of CFAP52 in male fertility and how can antibodies help investigate infertility phenotypes?

CFAP52 plays a critical role in male fertility through its functions in sperm head-tail connecting apparatus (HTCA) formation and flagellar development. Research using CFAP52 antibodies has revealed:

• CFAP52 localizes to both the HTCA and full-length flagella in normal sperm .

• CFAP52-deficient mice exhibit complete male sterility despite normal mating behavior .

• CFAP52 mutations in humans are associated with asthenoteratospermia, a condition characterized by reduced sperm motility and abnormal morphology .

• CFAP52 knockout mice display a mixed phenotype of abnormal sperm head-tail connection (acephalic sperm syndrome or ASS) and multiple morphological abnormalities of the flagella (MMAF) .

For investigating infertility phenotypes, researchers can use CFAP52 antibodies for:

• Comparative immunofluorescence studies between normal and patient sperm samples

• Analyzing CFAP52 protein expression levels in testicular biopsies

• Exploring CFAP52 localization patterns during spermatogenesis

• Screening infertile patients for CFAP52-related defects as a potential diagnostic biomarker

How can I verify the specificity of CFAP52 antibodies in my experimental system?

To verify CFAP52 antibody specificity:

• Positive and negative controls:

  • Use testis tissue from wild-type animals as a positive control

  • Include CFAP52-knockout tissue/cells as a negative control where available

  • Compare staining patterns with published CFAP52 localization data

• Western blot validation:

  • Confirm the detection of a single band at the expected molecular weight (~68.3 kDa for human CFAP52)

  • Test with recombinant CFAP52 protein alongside your experimental samples

  • If using transfected cells, compare FLAG-tagged CFAP52 expression with antibody detection

• Blocking peptide experiments:

  • Pre-incubate the CFAP52 antibody with the immunizing peptide

  • This should eliminate specific staining in your assay

• Cross-validation methods:

  • Use multiple antibodies targeting different epitopes of CFAP52

  • Confirm results with alternative techniques (e.g., mass spectrometry)

  • Compare results from antibody staining with mRNA expression data

What are the optimal methods for studying CFAP52 protein interactions using antibodies?

CFAP52 contains WD repeat domains that function as protein interaction scaffolds, making protein-protein interaction studies particularly relevant. The following methodologies are recommended:

• Co-immunoprecipitation (co-IP):

  • For exogenous expression: Transfect HEK293T cells with FLAG-tagged CFAP52 and Myc-tagged potential interacting proteins

  • For endogenous interactions: Use CFAP52 antibodies to immunoprecipitate native protein complexes from testis lysates

  • Western blot analysis can confirm interactions with candidate proteins

• Proximity ligation assay (PLA):

  • Useful for detecting in situ protein interactions

  • Requires antibodies raised in different species against CFAP52 and potential binding partners

• Immunofluorescence co-localization:

  • Use dual-labeling techniques to visualize spatial overlap between CFAP52 and candidate interacting proteins

  • Particularly informative in sperm and ciliated cells

Research has confirmed interactions between CFAP52 and multiple axonemal components including:

• SPATA6 (sperm head-tail connecting protein)

• CFAP45 (another microtubule inner protein)

• Components of outer dynein arms (DNAI1, DNAH11, DNAI2)

• Dynein regulatory complex components (DRC10)

• Radial spoke proteins (RSPH1, RSPH3)

• MIPs components (CFAP20, PACRG)

How do CFAP52 expression patterns differ between various ciliated tissues and what antibody approaches best capture these differences?

CFAP52 expression shows tissue-specific patterns with varying functional significance:

The research findings reveal interesting species-specific dependencies on CFAP52:

• In mice, CFAP52 deficiency causes both hydrocephalus and male infertility

• In humans, CFAP52 mutations have been associated primarily with male infertility without obvious ciliopathy phenotypes

For optimal detection of these tissue-specific patterns:

• Use tissue-specific fixation protocols (4% PFA for most applications)

• Employ antigen retrieval methods for IHC applications

• Consider dual-labeling with ciliary markers (acetylated tubulin) to confirm ciliary localization

• Use super-resolution microscopy for detailed subcellular localization studies

What methods can be used to study the effects of CFAP52 mutations on protein stability and function?

Researchers investigating CFAP52 mutations can employ several sophisticated approaches:

• Cycloheximide chase assays:

  • Treat cells expressing wild-type or mutant CFAP52 with cycloheximide to block protein synthesis

  • Monitor protein degradation rates over time (0-12 hours) by western blotting

  • This approach revealed that CFAP52 stabilizes its interacting partner SPATA6

• Expression analysis of mutant proteins:

  • Transfect cells with wild-type or mutant CFAP52 constructs

  • Western blotting can determine if nonsense mutations (e.g., p.W376*) result in truncated protein or complete absence of expression

  • Research has shown that the c.1128G>A/p.W376* mutation leads to undetectable CFAP52 expression

• Mini-gene splicing assays for splice-site mutations:

  • Clone genomic DNA fragments containing relevant exons and introns into a minigene vector

  • Transfect cells and analyze splicing patterns by RT-PCR and gel electrophoresis

  • This method demonstrated that the c.203G>T variant causes exon 2 skipping, resulting in a frameshift (p.D24Vfs*5)

• Functional rescue experiments:

  • Introduce wild-type CFAP52 into CFAP52-deficient cells or model organisms

  • Assess recovery of normal phenotype (e.g., sperm motility, axonemal structure)

  • This approach can confirm the pathogenicity of identified mutations

• Transmission electron microscopy (TEM):

  • Examine ultrastructural defects in axonemal structure in samples with CFAP52 mutations

  • Studies show that CFAP52 deficiency disrupts the "9+2" axonemal structure in sperm flagella

How can CFAP52 antibodies be used to investigate ciliopathies beyond male infertility?

While CFAP52 mutations have been primarily associated with male infertility, CFAP52 antibodies can be valuable tools for investigating other ciliopathies:

• Hydrocephalus research:

  • CFAP52-knockout mice develop hydrocephalus with sparse ependymal cilia

  • Immunostaining brain sections can reveal CFAP52 expression patterns in ependymal cells

  • Comparative studies between wild-type and disease models can elucidate CFAP52's role in cerebrospinal fluid circulation

• Primary ciliary dyskinesia (PCD) investigations:

  • Although tracheal cilia appear structurally normal in CFAP52-deficient mice , CFAP52 might still influence ciliary function

  • Combining immunofluorescence with high-speed video microscopy can assess both CFAP52 localization and ciliary beat patterns

• Heterotaxy studies:

  • CFAP52 has been associated with heterotaxy , a condition involving abnormal left-right organ arrangement

  • Antibody staining of embryonic nodal cilia can help investigate CFAP52's role in left-right patterning

For these applications, researchers should:

• Use tissue-specific controls

• Combine CFAP52 antibody staining with functional assays

• Consider species differences (human CFAP52 mutations may have tissue-specific effects different from mouse models)

What are the recommended protocols for using CFAP52 antibodies in diverse experimental techniques?

How can researchers effectively use CFAP52 antibodies for studying axonemal structure and function?

CFAP52 is a microtubule inner protein crucial for axonemal structure. Researchers can leverage CFAP52 antibodies for studying axoneme components through:

• High-resolution microscopy approaches:

  • Super-resolution techniques (STED, STORM) can precisely localize CFAP52 within the axonemal structure

  • Combine with other axonemal markers to create detailed maps of protein distributions

  • Immuno-electron microscopy can provide nanometer-scale localization data

• Comparative studies across mutant models:

  • Use CFAP52 antibodies to examine localization patterns in various axonemal mutants

  • This approach revealed that CFAP52 interacts with multiple axonemal components including:

    • Outer dynein arms and their docking complexes (DNAI1, DNAI2, DNAH11, ODAD1, ODAD3)

    • Microtubule inner proteins (CFAP45, CFAP20, PACRG)

    • Radial spoke components (RSPH1, RSPH3)

• Protein stability and interaction networks:

  • CFAP52 contains WD repeat domains that serve as interaction scaffolds

  • Antibody-based studies demonstrated that CFAP52 stabilizes SPATA6 and potentially other interacting partners

  • Co-immunoprecipitation followed by mass spectrometry can map the complete CFAP52 interactome

• Developmental studies:

  • Track CFAP52 expression during ciliogenesis or spermatogenesis

  • CFAP52 localizes to the manchette of elongating spermatids before redistributing to the mature flagellum

What approaches can help resolve contradictory findings in CFAP52 research using antibodies?

Research on CFAP52 has produced some contradictory findings, particularly regarding phenotypes in knockout models. Antibody-based approaches can help resolve these contradictions:

• Addressing differences in knockout phenotypes:

  • Two different CFAP52-knockout mouse models showed some phenotypic differences

  • One study reported disorganized junction of midpiece and principal piece with normal "9+2" axonemal structure

  • Another study showed disrupted "9+2" axonemal structures

  • Resolution approach: Use CFAP52 antibodies for detailed immunolocalization studies comparing both models directly

• Species-specific differences:

  • CFAP52-knockout mice exhibit hydrocephalus and male infertility

  • Human CFAP52 mutations appear to primarily affect fertility without obvious ciliopathy phenotypes

  • Resolution approach: Comparative immunostaining of human and mouse tissues to identify potential compensatory mechanisms

• Methodology standardization:

  • Create a standardized antibody validation protocol

  • Document exact experimental conditions including fixation methods, antibody dilutions, and incubation times

  • Perform direct comparisons of multiple CFAP52 antibodies on the same samples

• Collaborative cross-laboratory testing:

  • Exchange antibodies and protocols between laboratories reporting contradictory results

  • Use identical samples to determine if differences arise from technical variations or biological factors

The discrepancies might be due to:

• Different targeting strategies in gene knockout models

• Background strain effects

• Technical variations in sample preparation

• Variations in antibody specificity or sensitivity

How might CFAP52 antibodies contribute to developing diagnostic tools for male infertility?

CFAP52 antibodies show significant potential for developing diagnostic tools for male infertility based on recent findings:

• Clinical diagnostic applications:

  • CFAP52 mutations have been identified in patients with asthenoteratospermia (reduced sperm motility and abnormal morphology)

  • Antibody-based screening could identify patients with CFAP52-related defects

  • Immunofluorescence staining of patient sperm samples can detect abnormal or absent CFAP52 localization

• Development of diagnostic panels:

  • Create multiplexed immunofluorescence assays targeting CFAP52 alongside other proteins implicated in male infertility

  • This approach could help classify infertility subtypes based on molecular defects

  • The combined analysis of CFAP52 with its interacting partners (SPATA6, axonemal components) would provide comprehensive diagnostic information

• Non-invasive diagnostic approaches:

  • Develop techniques to detect CFAP52 abnormalities in semen samples without requiring testicular biopsies

  • Standardized immunofluorescence protocols could be implemented in fertility clinics

• Genetic screening correlation:

  • CFAP52 antibody-based diagnostics could complement genetic testing

  • Patients with abnormal CFAP52 protein expression or localization would be candidates for CFAP52 gene sequencing

The study reveals that CFAP52 might serve as a novel diagnostic target specifically for male infertility characterized by head-tail connection defects and flagellar abnormalities .

What novel experimental techniques could enhance CFAP52 antibody-based research?

Several cutting-edge techniques could significantly advance CFAP52 antibody-based research:

• Expansion microscopy:

  • Physical expansion of specimens can provide super-resolution imaging using standard microscopes

  • This would allow detailed visualization of CFAP52 within the complex axonemal structure

  • Particularly valuable for examining the precise localization within the 9+2 axoneme architecture

• Live-cell imaging with nanobodies:

  • Develop CFAP52-specific nanobodies (single-domain antibodies)

  • Fuse with fluorescent proteins for live-cell tracking of CFAP52 dynamics

  • This approach could reveal temporal aspects of CFAP52 function during ciliogenesis

• Proximity-dependent biotinylation (BioID or TurboID):

  • Fuse CFAP52 with a biotin ligase

  • Identify proximal proteins in living cells

  • This would provide a comprehensive map of the CFAP52 interactome under various conditions

• Single-molecule tracking:

  • Use quantum dot-conjugated antibodies to track individual CFAP52 molecules

  • Reveal transport mechanisms and dynamics within the intraflagellar transport system

• Cryo-electron tomography with immunogold labeling:

  • Combine structural studies with precise localization of CFAP52

  • Create high-resolution 3D models of CFAP52's position within the axonemal structure

• CRISPR-based knockin of epitope tags:

  • Generate endogenously tagged CFAP52 in model organisms

  • Enable consistent antibody detection without overexpression artifacts

  • Facilitate precise developmental and tissue-specific studies

Implementing these advanced techniques would significantly enhance our understanding of CFAP52's role in ciliary and flagellar biology.

What are the most significant research findings about CFAP52 that researchers should be aware of?

The most significant CFAP52 research findings that researchers should be familiar with include:

• Structural role in axoneme:

  • CFAP52 functions as a microtubule inner protein (MIP) within the dynein-decorated doublet microtubules

  • It contributes to the stability of the B-tubules in the axonemal structure

• Critical role in male fertility:

  • CFAP52 is essential for proper sperm head-tail connection and flagellar formation

  • CFAP52-deficient mice are completely sterile despite normal mating behavior

  • Human mutations in CFAP52 are associated with asthenoteratospermia

• Protein interactions:

  • CFAP52 contains WD repeat domains that serve as protein interaction scaffolds

  • It interacts with and stabilizes SPATA6, a key structural protein of the head-tail connecting apparatus

  • CFAP52 also interacts with multiple axonemal components including outer dynein arms, radial spokes, and other MIPs

• Species-specific phenotypic differences:

  • Mouse models show both hydrocephalus and male infertility

  • Human patients with CFAP52 mutations primarily exhibit fertility issues without other obvious ciliopathies

• Molecular mechanisms:

  • CFAP52 regulates protein stability of its interacting partners

  • The degradation rate of SPATA6 is significantly slowed in the presence of CFAP52

These findings collectively position CFAP52 as an important target for male infertility research and highlight its complex roles in ciliary and flagellar biology.

How can researchers contribute to advancing CFAP52 antibody development and applications?

Researchers can advance CFAP52 antibody technology and applications through several approaches:

• Antibody characterization and validation:

  • Perform comprehensive validation using CFAP52-knockout tissues as negative controls

  • Compare multiple commercial antibodies to identify those with highest specificity and sensitivity

  • Document optimal conditions for various applications and share detailed protocols

• Development of application-specific antibodies:

  • Generate conformation-specific antibodies that recognize native CFAP52

  • Develop phospho-specific antibodies if CFAP52 regulation involves phosphorylation

  • Create epitope-mapped antibodies targeting different regions of CFAP52

• Cross-disciplinary collaborations:

  • Form partnerships between reproductive biologists, ciliary researchers, and clinical andrologists

  • Establish biobanks of patient samples with characterized CFAP52 mutations

  • Create standardized protocols for diagnostic applications

• Technological innovations:

  • Develop high-throughput screening methods using CFAP52 antibodies

  • Create multiplexed detection systems for CFAP52 and interacting partners

  • Implement machine learning algorithms for automated analysis of CFAP52 localization patterns

• Clinical translation:

  • Establish correlations between CFAP52 antibody staining patterns and fertility outcomes

  • Develop simplified protocols suitable for clinical diagnostic laboratories

  • Create reference standards for normal versus abnormal CFAP52 expression/localization