CFAP157 Antibody

What is the optimal sample preparation method for detecting CFAP157 in tissue samples?

For optimal detection of CFAP157 in tissue samples, fixation with 4% paraformaldehyde is recommended, followed by antigen retrieval using citrate buffer (pH 6.0). Since CFAP157 is primarily expressed in ciliated tissues including cervix, lung, testis, and uterus, these tissues should be sectioned at 4-5μm thickness for immunohistochemistry . When working with sperm samples, a gentle fixation protocol is critical to preserve flagellar structures where CFAP157 localizes to basal bodies. For immunofluorescence applications, use 0.25-2 μg/mL of antibody, while for immunohistochemistry, a dilution range of 1:1000-1:2500 typically yields optimal results .

How can I validate the specificity of my CFAP157 antibody?

Validating CFAP157 antibody specificity requires a multi-faceted approach:

• Orthogonal validation: Compare antibody staining patterns with RNA-seq data for CFAP157 expression across tissues .

• Independent antibody validation: Compare staining patterns using two independent antibodies targeting non-overlapping epitopes of CFAP157 .

• siRNA knockdown: Assess decreased staining intensity after CFAP157 downregulation .

• Biological validation: Confirm stronger signals in tissues known to express CFAP157 (testis, lung, ciliated epithelium) versus low-expression tissues .

• Western blot: Verify detection of a band at approximately 60.5 kDa (the predicted molecular weight of CFAP157) .

A comprehensive validation should demonstrate consistent results across multiple methods to ensure reliable experimental outcomes.

What are the key experimental controls needed when studying CFAP157 localization in ciliated tissues?

When designing experiments to study CFAP157 localization in ciliated tissues, the following controls are essential:

• Positive tissue controls: Include known CFAP157-expressing tissues such as testis and respiratory epithelium .

• Negative tissue controls: Include tissues with minimal CFAP157 expression.

• Antibody controls: Include a secondary-only control and an isotype control.

• Co-localization markers: Include antibodies against established basal body/centrosomal markers (e.g., CEP350) and axonemal markers (e.g., acetylated tubulin) to confirm CFAP157's subcellular localization .

• FOXJ1 expression correlation: Since CFAP157 is a downstream target of FOXJ1, correlating CFAP157 expression with FOXJ1 expression provides additional validation .

These controls help distinguish specific CFAP157 signals from background and confirm its reported subcellular localization at basal bodies.

How does CFAP157 protein interact with the centrosomal-ciliary interface, and what methodologies best capture these interactions?

CFAP157 protein localizes to basal bodies and interacts with tubulin and the centrosomal protein CEP350 . To effectively study these interactions:

• Proximity ligation assays (PLA): This technique can visualize protein-protein interactions between CFAP157 and its binding partners (CEP350, tubulin) with subcellular resolution.

• Co-immunoprecipitation followed by mass spectrometry: This approach can identify the complete interactome of CFAP157 at the centrosomal-ciliary interface.

• Super-resolution microscopy: Techniques such as STORM or STED can resolve the precise localization of CFAP157 relative to basal body structures.

• FRAP (Fluorescence Recovery After Photobleaching): This can assess the dynamics of CFAP157 association with the basal body.

Research indicates that CFAP157 functions at the centrosome/basal body during flagellum morphogenesis and may regulate the suppression of supernumerary axonemes . Understanding these interactions is crucial for elucidating the molecular mechanisms underlying the phenotypes observed in Cfap157-deficient models.

What are the most effective approaches for studying CFAP157 function in the context of male fertility research?

Based on research showing that Cfap157 knockout mice exhibit male infertility due to specific sperm motility defects , the following methodological approaches are recommended:

• Conditional knockout models: Generate tissue-specific knockouts to distinguish between developmental versus functional roles of CFAP157.

• Advanced sperm motility analysis: Use Computer-Assisted Sperm Analysis (CASA) combined with high-speed videomicroscopy to quantify specific motility parameters affected by CFAP157 deficiency.

• Electron microscopy: Transmission electron microscopy is essential for visualizing ultrastructural defects in sperm flagella, including the reported axonemal loops and supernumerary axonemes .

• Proteomic analysis: Compare the proteome of wild-type versus Cfap157-deficient sperm to identify downstream molecular pathways affected.

• Rescue experiments: Attempt to rescue fertility phenotypes through targeted expression of CFAP157 in knockout models.

The unique phenotype observed in Cfap157-deficient sperm (axonemal loops, supernumerary axonemes, and ectopic accessory structures) suggests a specific role in maintaining proper flagellar architecture during spermiogenesis .

How can CFAP157 antibodies be used to investigate ciliogenesis pathways across different motile ciliated tissues?

To investigate ciliogenesis pathways using CFAP157 antibodies:

• Developmental time-course studies: Analyze CFAP157 expression during different stages of ciliogenesis in various tissues (respiratory epithelium, ependymal cells, fallopian tube).

• Comparative analysis across tissues: Compare CFAP157 localization and expression patterns between different motile ciliated tissues to identify tissue-specific differences in ciliogenesis pathways.

• Co-expression analysis with FOXJ1: Since CFAP157 is a FOXJ1 target, analyze their temporal relationship during ciliogenesis .

• Primary cell culture models: Use air-liquid interface cultures of human airway epithelial cells to study CFAP157 dynamics during ciliogenesis in vitro.

• CRISPR-Cas9 genetic manipulation: Create cellular models with tagged endogenous CFAP157 to track its dynamics during ciliogenesis without overexpression artifacts.

CFAP157 is specifically expressed in motile ciliated tissues in a FOXJ1-dependent manner, making it a valuable marker for studying the transcriptional program of motile ciliogenesis across different tissues .

What are the common technical challenges when using CFAP157 antibodies in immunofluorescence studies of sperm, and how can they be addressed?

Working with CFAP157 antibodies in sperm samples presents several technical challenges:

Additionally, when imaging, use confocal microscopy with z-stacks to fully capture the 3D organization of CFAP157 along the flagellar structures. The antibody concentration should be optimized specifically for sperm samples, which may require higher dilutions (1:500-1:2000) than those used for tissue sections .

How can researchers distinguish between specific and non-specific binding when using CFAP157 antibodies in tissues with low expression levels?

Distinguishing specific from non-specific binding is particularly challenging in tissues with low CFAP157 expression. Implement these methodological approaches:

• Titration series: Test multiple antibody dilutions (1:200, 1:500, 1:1000, 1:2500) to identify the optimal signal-to-noise ratio .

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide to block specific binding sites.

• RNA-based validation: Correlate antibody staining with RT-qPCR or RNA-seq data for CFAP157 across tissues .

• Knockout validation: If available, use tissues from Cfap157 knockout models as negative controls .

• Sequential immunolabeling: Compare staining patterns when the primary antibody is applied before versus after the secondary antibody.

• Alternative fixation methods: Compare results using different fixatives to identify potential fixation artifacts.

For tissues with known low expression levels, consider using signal amplification methods such as tyramide signal amplification, but be aware that these methods may also amplify background signals.

How should researchers design experiments to investigate the relationship between CFAP157 and FOXJ1 in motile ciliogenesis?

To effectively study the CFAP157-FOXJ1 relationship in motile ciliogenesis:

• Temporal expression analysis: Use time-course experiments during ciliogenesis to determine the order of FOXJ1 and CFAP157 expression.

• ChIP-seq analysis: Confirm direct binding of FOXJ1 to the CFAP157 promoter region.

• Reporter assays: Create CFAP157 promoter constructs with wild-type and mutated FOXJ1 binding sites to test direct regulation.

• Rescue experiments: Test whether CFAP157 expression can rescue specific aspects of the FOXJ1 knockout phenotype.

• Single-cell RNA-seq: Analyze co-expression patterns of FOXJ1 and CFAP157 during different stages of ciliogenesis.

Research has shown that CFAP157 is specifically expressed in motile ciliated tissues in mouse and Xenopus in a FOXJ1-dependent manner . Understanding this relationship is crucial for elucidating the transcriptional network governing motile ciliogenesis.

What methodological approaches should be used to accurately compare CFAP157 expression across different tissue samples?

When comparing CFAP157 expression across different tissues, several methodological considerations are essential:

• Standardized sample preparation: Use consistent fixation protocols and processing times across all samples.

• Batch processing: Process all samples simultaneously with the same antibody lot and reagents to minimize technical variation.

• Internal controls: Include positive control tissues (testis, respiratory epithelium) in each batch .

• Quantification methods: Use digital image analysis with appropriate thresholding to quantify staining intensity.

• Normalization strategies: Normalize CFAP157 expression to the proportion of ciliated cells in each tissue.

For quantitative comparisons, consider these advanced approaches:

• Multiplex immunofluorescence: Co-stain with ciliated cell markers to normalize CFAP157 expression to ciliated cell numbers.

• RNA-seq validation: Correlate protein expression with transcript levels.

• Multiple antibodies: Use different antibodies targeting different CFAP157 epitopes to confirm expression patterns .

CFAP157 is expressed in various human tissues including cervix, lung, testis, and uterus, but expression levels vary significantly .

How can researchers integrate structural information about CFAP157 protein with antibody epitope selection for optimal detection in different applications?

For optimal detection of CFAP157 across different applications, researchers should integrate structural information with epitope selection:

• Structural analysis: CFAP157 is primarily composed of α-helices with no transmembrane domains . Target antibody epitopes within predicted surface-exposed regions.

• Epitope accessibility analysis: The immunogen sequence used in commercial antibodies (e.g., "DNQALKSQRDQLSLQLEQQQVDLQRLQQELANEQKVRASLEAALVQATSFLQNILQMHRDEEDSDVDVTFQPWHKEMLQQLLVMLSST") should be evaluated for accessibility in native protein conformations .

• Application-specific considerations:

  • For Western blot: Choose epitopes that remain accessible in denatured conditions.

  • For immunoprecipitation: Select epitopes not involved in protein-protein interactions.

  • For immunofluorescence: Target epitopes accessible in fixed but non-denatured conditions.

• Computational epitope prediction: Use biophysics-informed models to predict epitope accessibility and immunogenicity .

When selecting commercial antibodies, consider those with documented validation in your specific application. The antibody targeting amino acids 1-200 may be optimal for certain applications, while C-terminal antibodies may be better for others based on the protein's structural features .

What are the methodological considerations for investigating potential roles of CFAP157 in human ciliopathies?

To investigate CFAP157's potential involvement in human ciliopathies, researchers should consider:

• Cohort selection: Focus on male infertility cases with specific sperm flagellar defects similar to those observed in Cfap157 knockout mice .

• Genetic screening approach: Use targeted sequencing of CFAP157 in selected patient cohorts rather than whole-exome sequencing for better coverage.

• Functional validation: For identified variants, use in vitro models to assess impact on:

  • Protein localization to basal bodies

  • Interaction with known partners (tubulin, CEP350)

  • Ability to rescue the phenotype in Cfap157-deficient cellular models

• Mouse models: Generate knock-in mice harboring human variants to assess phenotypic impacts in vivo.

• Tissue-specific considerations: Analyze both sperm and respiratory epithelium samples from patients with suspected CFAP157 mutations.

Given that CFAP157 knockout mice show male infertility due to specific sperm flagellar defects , human variants might contribute to similar phenotypes or potentially broader ciliopathy syndromes affecting multiple ciliated tissues.

How can advanced antibody design principles be applied to create more specific and sensitive CFAP157 antibodies for challenging research applications?

For developing next-generation CFAP157 antibodies with enhanced specificity and sensitivity:

• Biophysics-informed modeling: Implement computational approaches that identify distinct binding modes and predict antibody-antigen interactions .

• Epitope mapping optimization: Target unique regions of CFAP157 with minimal homology to other proteins, particularly focusing on regions that distinguish it from other centrosomal/ciliary proteins.

• Phage display selection: Perform selections against various combinations of CFAP157 domains to identify antibodies with customized specificity profiles .

• Cross-species reactivity design: Engineer antibodies that recognize conserved epitopes across species to facilitate translational research.

• Application-specific optimization: Design separate antibodies optimized for Western blot, immunoprecipitation, or immunohistochemistry based on epitope accessibility in different conditions.

Advanced antibody design can leverage biophysics-informed models to identify binding modes associated with specific ligands, enabling the prediction and generation of antibody variants with customized specificity profiles . This approach has particular value for studying CFAP157 in complex ciliary structures where cross-reactivity with other components can complicate interpretation.