DNAH7 Antibody

What is DNAH7 and what is its biological significance?

DNAH7 (Dynein, Axonemal, Heavy Chain 7) is an axonemal dynein heavy chain protein that forms part of the inner dynein arms (IDAs) in ciliary structures. It has a molecular weight of approximately 461 kDa and is uniquely positioned as a component of both IDAb and IDAe complexes within the ciliary axoneme . DNAH7 is associated with the light intermediate chain DNALI1 and plays a crucial role in ciliary motility. The protein is localized throughout the entire length of ciliary axonemes in healthy individuals, and its absence or mislocalization is associated with ciliary dysfunction disorders . Unlike other dynein heavy chains that appear in a single IDA type, human DNAH7 is found in two different single-headed IDA types, making it an important structural and functional component of the ciliary machinery.

What types of DNAH7 antibodies are available for research?

Several types of DNAH7 antibodies are available for research applications, including:

• Polyclonal antibodies: Most commonly from rabbit hosts, these recognize different epitopes of the DNAH7 protein .

• Region-specific antibodies: Antibodies targeting specific amino acid regions (e.g., AA 2301-2400 or AA 730-903) of the DNAH7 protein .

• Conjugated antibodies: Available with various conjugates including:

  • Unconjugated forms for flexible detection strategies

  • HRP-conjugated for enzymatic detection

  • FITC-conjugated for fluorescence applications

  • Biotin-conjugated for amplification systems

The specificity of these antibodies has been validated through Western blot analysis, confirming the detection of a single band at the expected molecular weight of DNAH7 (~461 kDa) .

What are the common applications for DNAH7 antibodies?

DNAH7 antibodies are utilized across multiple research applications, particularly in ciliary function studies:

Immunofluorescence (IF) microscopy with DNAH7 antibodies has become an important diagnostic tool for characterizing ciliary defects, particularly in primary ciliary dyskinesia (PCD) research . These antibodies are particularly valuable when used in conjunction with other ciliary markers such as acetylated α-tubulin to examine co-localization patterns.

How do defects in the 96 nm axonemal ruler affect DNAH7 localization?

Research has demonstrated that defects in the 96 nm axonemal ruler components, specifically mutations in CCDC39 and CCDC40 genes, directly impact the localization of DNAH7 in respiratory ciliary axonemes . In individuals with disease-causing variants in either CCDC39 or CCDC40, immunofluorescence studies show complete absence of DNAH7 from the ciliary axoneme, in contrast to its normal distribution throughout the entire ciliary length in healthy controls .

This absence is specific to ruler defects, as PCD individuals with other types of defects (such as N-DRC defects caused by variants in DRC1/CCDC164, CCDC65, or GAS8, or ODA defects caused by DNAH5 variants) display normal DNAH7 distribution along the ciliary axoneme . This selective impact demonstrates the critical role of the 96 nm ruler structure in proper assembly and integration of DNAH7 into the axonemal architecture, providing insights into the hierarchical assembly process of ciliary components.

What is the relationship between DNAH7 and other inner dynein arm components?

DNAH7 maintains specific associations with other IDA components that are critical for proper ciliary function:

• DNAH7 is uniquely positioned as a component of both IDAb and IDAe complexes, making it the only dynein heavy chain in humans to appear in two different single-headed IDA types .

• DNAH7 is functionally associated with DNALI1 (Dynein Axonemal Light Intermediate Chain 1), and both proteins are affected in similar patterns in ciliary dyskinesia disorders .

• Unlike DNAH6 (which is associated with CETN2 in IDAg), DNAH7's association with DNALI1 represents a distinct assembly pathway for IDA components .

• When analyzing ciliary defects, the pattern of DNAH7 mislocalization often parallels that of DNALI1, providing a useful diagnostic correlation .

Understanding these relationships is crucial for comprehending the complex assembly and function of inner dynein arms in human ciliary structures.

How can DNAH7 antibodies contribute to understanding ciliopathy mechanisms?

DNAH7 antibodies provide valuable tools for investigating the molecular mechanisms underlying ciliopathies, particularly Primary Ciliary Dyskinesia (PCD):

• Differential diagnosis: By examining DNAH7 localization patterns in respiratory cilia from patients with suspected ciliopathies, researchers can distinguish between different molecular causes of ciliary dysfunction .

• Genotype-phenotype correlations: DNAH7 staining patterns can be correlated with specific genetic mutations, helping to establish clearer connections between genotype and cellular phenotype in ciliopathies .

• Hierarchical assembly mapping: The absence of DNAH7 in patients with ruler protein defects (CCDC39/CCDC40) but not in patients with N-DRC or ODA defects helps map the hierarchical dependencies in ciliary assembly processes .

• Functional domain analysis: By using antibodies targeting different epitopes of DNAH7, researchers can investigate which domains are critical for proper localization and function within the axoneme .

These applications collectively contribute to a deeper understanding of ciliary biology and the pathogenesis of ciliopathies.

What are the best practices for immunofluorescence detection of DNAH7?

For optimal immunofluorescence detection of DNAH7 in ciliary structures, consider the following methodological recommendations:

• Sample preparation:

  • For respiratory epithelial cells, brush biopsies or nasal scrapes provide suitable material

  • Immediate fixation (typically 4% paraformaldehyde) is critical to preserve axonemal structure

  • For paraffin sections, antigen retrieval steps are essential (dilution 1:50-200)

• Antibody selection and validation:

  • Verify antibody specificity using Western blot analysis (expected band at ~461 kDa)

  • Use proper controls including known positive samples and isotype controls

  • For co-localization studies, pair DNAH7 antibodies with established ciliary markers such as acetylated α-tubulin

• Imaging considerations:

  • Confocal microscopy is preferred for detailed localization studies

  • Z-stack acquisition ensures comprehensive visualization of the three-dimensional ciliary structure

  • Quantitative analysis of signal intensity along the length of cilia provides objective assessment of DNAH7 distribution

The recommended dilution for immunofluorescence applications ranges from 1:50-200, with adjustments based on the specific antibody and sample preparation method .

How can Western blot analysis be optimized for DNAH7 detection?

Western blot analysis of DNAH7 presents unique challenges due to its high molecular weight (~461 kDa). The following protocol modifications are recommended:

• Sample preparation:

  • Use isolated ciliary axonemes when possible for enriched DNAH7 content

  • Include protease inhibitors to prevent degradation of this large protein

  • Extended sonication may be necessary to ensure complete protein extraction

• Gel electrophoresis considerations:

  • Use low percentage (3-5%) polyacrylamide gels or gradient gels

  • Extended running time at lower voltage improves separation of high molecular weight proteins

  • Special ladder markers that include proteins >400 kDa should be used

• Transfer optimization:

  • Wet transfer is recommended over semi-dry methods

  • Longer transfer times (overnight at low amperage) improve transfer efficiency

  • Reduced methanol concentration in transfer buffer facilitates movement of large proteins

• Detection recommendations:

  • Primary antibody dilution of 1:500-1000 is typically effective

  • Enhanced chemiluminescence with extended exposure times may be necessary

  • Use of fluorescently-labeled secondary antibodies can provide better quantification

Western blot analysis has successfully confirmed DNAH7 as an axonemal component of respiratory cilia by detecting a specific band at the expected size of approximately 461 kDa .

What controls should be included when studying DNAH7 in ciliopathy research?

Proper experimental controls are essential when using DNAH7 antibodies in ciliopathy research:

• Positive controls:

  • Respiratory epithelial cells from healthy donors showing normal DNAH7 localization throughout ciliary axonemes

  • Known functional axonemal preparations with confirmed DNAH7 expression

• Negative controls:

  • Primary antibody omission to assess background staining

  • Isotype controls to evaluate non-specific binding

  • When available, samples from individuals with confirmed DNAH7 mutations

• Disease-specific controls:

  • Samples from individuals with CCDC39/CCDC40 mutations (expected to show DNAH7 absence)

  • Samples from individuals with N-DRC defects or ODA defects (expected to show normal DNAH7 localization)

  • This comparative approach helps distinguish ruler-specific effects from other ciliary defects

• Technical validation:

  • Cross-validation using multiple antibodies targeting different DNAH7 epitopes

  • Complementary techniques such as Western blot analysis to confirm antibody specificity

  • RT-PCR to assess DNAH7 transcript expression when appropriate

Inclusion of these controls ensures reliable interpretation of DNAH7 staining patterns in experimental and diagnostic applications.

How can DNAH7 antibodies be used in the diagnosis of Primary Ciliary Dyskinesia?

DNAH7 antibodies have emerging utility in the diagnostic approach to Primary Ciliary Dyskinesia (PCD):

• Differential diagnostics:

  • DNAH7 immunofluorescence patterns help distinguish between different molecular subtypes of PCD

  • The absence of DNAH7 in ciliary axonemes is specifically associated with defects in the 96 nm ruler (CCDC39/CCDC40 mutations)

  • Normal DNAH7 localization despite other ciliary defects points to different molecular causes (such as N-DRC or ODA defects)

• Complementary diagnostic approach:

  • DNAH7 staining should be used alongside other established markers including DNALI1, DNAH1, and DNAH6

  • The combined staining pattern provides a comprehensive assessment of IDA integrity

  • This multi-marker approach increases diagnostic sensitivity and specificity

• Integration with other diagnostic modalities:

  • Correlate DNAH7 staining results with transmission electron microscopy (TEM) findings

  • Combine with high-speed video microscopy analysis (HVMA) of ciliary beat patterns

  • Support molecular genetic testing by narrowing down candidate genes based on specific staining patterns

The combined use of antibodies directed against DNAH1, DNAH6, and DNAH7 in immunofluorescence microscopy provides powerful tools for the detailed characterization of ciliary defects in suspected PCD cases .

What are the limitations of DNAH7 antibody-based diagnostics?

Despite their utility, DNAH7 antibody-based approaches have several important limitations that researchers should consider:

• Specificity challenges:

  • Cross-reactivity with other dynein heavy chains may occur due to structural similarities

  • Validation through Western blot analysis is essential to confirm antibody specificity

  • Different epitope-targeting antibodies may yield slightly different localization patterns

• Technical considerations:

  • Sample quality is critical; degraded samples may yield false-negative results

  • Proper fixation and processing protocols must be strictly followed

  • Interpretation requires experienced personnel familiar with normal ciliary architecture

• Diagnostic limitations:

  • DNAH7 absence is not specific to a single gene defect but rather to a category of defects (ruler protein dysfunction)

  • Not all PCD subtypes will show DNAH7 abnormalities

  • Interpretation should always be in context with other clinical, ultrastructural, and genetic findings

• Research gaps:

  • The exact functional significance of DNAH7 in different IDA positions is not fully understood

  • Limited studies on DNAH7 in non-respiratory cilia restrict broader application

  • More research is needed on the relationship between DNAH7 mislocalization and clinical phenotypes

Awareness of these limitations helps researchers appropriately design experiments and interpret results when using DNAH7 antibodies in both research and diagnostic contexts.

What are promising research applications for DNAH7 antibodies beyond ciliopathy diagnostics?

DNAH7 antibodies hold potential for expanding research beyond traditional ciliopathy diagnostics:

• Developmental biology studies:

  • Investigation of DNAH7 expression during ciliogenesis

  • Examination of tissue-specific differences in DNAH7 incorporation into axonemes

  • Analysis of DNAH7 role in embryonic development, particularly in processes dependent on ciliary motility

• Therapeutic development:

  • Screening potential compounds that could correct DNAH7 mislocalization

  • Monitoring treatment efficacy in gene therapy approaches targeting ciliary assembly

  • Assessing pharmacological interventions aimed at improving ciliary function

• Model system validation:

  • Verification of DNAH7 conservation and function in animal models of ciliopathies

  • Validation of in vitro ciliated cell models for high-throughput screening

  • Evaluation of patient-derived organoid systems for personalized medicine approaches

• Structural biology applications:

  • Using super-resolution microscopy with DNAH7 antibodies to map precise axonemal architecture

  • Correlative light and electron microscopy to relate DNAH7 positioning to ultrastructural features

  • Integration with cryo-EM studies to refine structural models of dynein arm complexes

These expanding applications demonstrate the versatility of DNAH7 antibodies as tools for advancing our understanding of fundamental ciliary biology and ciliopathy pathogenesis.