DNAH5 Antibody

What is DNAH5 and why is it important in ciliary research?

DNAH5 (Dynein Axonemal Heavy Chain 5) is a critical force-generating protein of respiratory cilia that produces force toward the minus ends of microtubules. It possesses ATPase activity, with the force-producing power stroke occurring upon ADP release. DNAH5 is essential for the structural and functional integrity of ependymal cell cilia lining brain ventricles. Its significance stems from being the most frequently mutated gene in primary ciliary dyskinesia (PCD), making it a crucial target for understanding ciliopathies . When studying ciliary dysfunction, DNAH5 antibodies serve as vital tools for assessing the presence, localization, and structural integrity of this protein within the ciliary axoneme.

How do I select the most appropriate DNAH5 antibody for my research application?

Selecting the optimal DNAH5 antibody requires careful consideration of several factors:

• Application compatibility: Different experimental techniques require antibodies validated for specific applications. Based on the search results, DNAH5 antibodies are available for multiple applications:

  • Immunohistochemistry (IHC/IHC-P): For tissue sections and paraffin-embedded samples

  • Immunofluorescence (IF): For cellular localization studies

  • Western blot (WB): For protein expression analysis

  • ELISA: For quantitative detection

  • Flow cytometry (FCM): For cellular analysis

• Species reactivity: Verify the antibody's reactivity with your experimental model. Most DNAH5 antibodies are reactive with human samples, while some also detect mouse and rat orthologs .

• Immunogen region: Consider antibodies targeting different epitopes of DNAH5. For example, ab122390 targets the region within amino acids 900-1050, while another antibody (ab234826) has been successfully used in PCD research .

• Validation evidence: Prioritize antibodies with published citations, especially those used in similar research contexts. The antibody ab234826 has been used in DNAH5-mutated PCD airway organoid research, suggesting its reliability for PCD studies .

What structural characteristics of DNAH5 protein are important to consider when selecting an antibody?

DNAH5 is a large protein (approximately 529 kDa) with distinct functional domains that require consideration when selecting antibodies:

• Size consideration: The significant molecular weight of DNAH5 (529 kDa) requires special attention during Western blot analysis, including the use of specialized gels (3-8% Tris-acetate) for proper separation .

• Functional domains: DNAH5 contains multiple functional regions, including ATP-binding domains and microtubule-binding regions. Antibodies targeting conserved functional domains might provide more consistent results across species.

• Mutation hotspots: In PCD research, consider antibodies that target regions distinct from common mutation sites, unless your specific goal is to detect mutant variants. Novel pathogenic variants have been identified throughout the DNAH5 gene, including missense mutations like c.12614G>A .

• Post-translational modifications: Consider whether your research requires detection of specific post-translationally modified forms of DNAH5, as these modifications might affect antibody binding.

What are the optimal protocols for DNAH5 immunostaining in ciliated tissues?

Based on published methodologies for DNAH5 immunostaining in ciliated tissues:

For section immunofluorescence:

• Harvest and fix tissues or organoids in 4% paraformaldehyde at 4°C overnight

• Dehydrate, embed in paraffin, and section the samples

• Perform antigen retrieval (if recommended for your specific antibody)

• Block with 5% bovine serum albumin (BSA) for 1 hour at room temperature

• Incubate with primary DNAH5 antibody (e.g., ab234826) at 4°C overnight

• Wash and incubate with appropriate fluorophore-conjugated secondary antibody

• Counterstain and mount for visualization

For whole-mount immunofluorescence:

• Fix samples in 4% paraformaldehyde at 4°C overnight

• Permeabilize using 0.5% Triton X-100

• Block with 5% BSA for 1 hour at room temperature

• Incubate with DNAH5 antibody at 4°C overnight

• Proceed with secondary antibody incubation and imaging

Co-staining with acetylated tubulin (marker for cilia) and other ciliary components provides valuable context for DNAH5 localization.

How can I optimize Western blot protocols for detecting the large DNAH5 protein?

DNAH5's large size (529 kDa) presents challenges for Western blot analysis that require specific optimization:

• Sample preparation:

  • Lyse samples in ice-cold RIPA buffer containing protease inhibitor cocktail

  • Perform ultrasonic disruption to ensure complete protein extraction

  • Incubate on ice for approximately 40 minutes

  • Add 5× SDS loading buffer and heat at 95°C for denaturation

• Gel selection and electrophoresis:

  • Use specialized 3-8% Tris-acetate gels designed for high molecular weight proteins

  • Run at lower voltage for extended periods to achieve proper separation

• Transfer optimization:

  • Transfer to 0.45-μm pore size PVDF membranes (not 0.2-μm)

  • Perform overnight transfer at low voltage (40V) to ensure complete transfer of large proteins

• Detection considerations:

  • Include positive controls and molecular weight markers appropriate for high molecular weight proteins

  • Use GAPDH or other housekeeping proteins as loading controls

  • Consider enhanced chemiluminescence systems with extended exposure times for optimal signal detection

What control samples should be included when validating DNAH5 antibody specificity?

Robust validation of DNAH5 antibody specificity requires several controls:

• Positive tissue controls:

  • Human respiratory epithelium (bronchial or nasal) with known DNAH5 expression

  • Fallopian tube tissue (shown to express DNAH5)

  • Normal airway organoids (compared to DNAH5-mutated organoids)

• Negative controls:

  • Primary antibody omission controls

  • Tissues lacking ciliated cells

  • DNAH5-knockout or DNAH5-mutated samples (such as PCD patient samples with confirmed DNAH5 mutations)

• Specificity controls:

  • Peptide competition assays using the immunogen peptide

  • Comparison with alternative DNAH5 antibodies targeting different epitopes

  • siRNA knockdown of DNAH5 in appropriate cell lines

• Cross-reactivity assessment:

  • Testing on tissues from multiple species when using antibodies claimed to have cross-species reactivity

  • Checking for signal in tissues known to express DNAH5 homologs or related dynein proteins

Why might DNAH5 antibody staining show unexpected localization patterns in ciliated cells?

Unexpected DNAH5 localization patterns may result from various technical and biological factors:

• Technical factors:

  • Inadequate fixation: Overfixation can mask epitopes while underfixation can disrupt structural integrity

  • Insufficient permeabilization: DNAH5 is an intracellular protein requiring adequate permeabilization

  • Non-specific binding: Increase blocking duration or concentration (5% BSA recommended)

  • Antibody concentration: Titrate primary antibody to find optimal concentration

• Biological factors:

  • DNAH5 mutations: In PCD patients, DNAH5 may be absent from the axoneme or show abnormal distribution

  • Developmental stage: DNAH5 expression and localization may vary during ciliogenesis

  • Cell type heterogeneity: Different ciliated cell populations may exhibit varying DNAH5 patterns

  • Ciliary damage: Environmental factors or sample processing can damage cilia, altering DNAH5 distribution

• Validation approaches:

  • Co-stain with acetylated tubulin to confirm ciliary structures

  • Compare with transmission electron microscopy (TEM) analysis to correlate with outer dynein arm (ODA) presence/absence

  • Use multiple antibodies targeting different DNAH5 epitopes

How can I differentiate between non-specific binding and true DNAH5 signal in immunofluorescence studies?

Distinguishing between specific and non-specific signals requires systematic controls and analysis:

• Control implementations:

  • Primary antibody omission control to assess secondary antibody non-specific binding

  • Include DNAH5-deficient samples (from PCD patients with confirmed DNAH5 mutations)

  • Perform peptide competition assays with immunizing peptide

  • Use isotype control antibodies matched to your primary antibody

• Signal pattern analysis:

  • Specific DNAH5 signal should localize to ciliary axonemes in wild-type samples

  • Compare to established patterns: DNAH5 normally localizes along the entire length of cilia

  • In PCD patients with DNAH5 mutations, the protein is typically absent from the axoneme or present only in the proximal region

• Co-localization assessment:

  • Co-stain with established ciliary markers (acetylated tubulin, other ODA components)

  • True DNAH5 signal should show consistent co-localization with other ODA components in wild-type samples

What are the critical parameters for optimizing immunoprecipitation (IP) of DNAH5?

Immunoprecipitation of DNAH5 presents challenges due to its large size and complex structure:

• Lysis buffer optimization:

  • Use gentle, non-denaturing buffers that maintain protein-protein interactions

  • Include protease inhibitors to prevent degradation

  • Consider adding phosphatase inhibitors if studying DNAH5 phosphorylation

  • Optimize detergent concentration to solubilize membrane-associated DNAH5 without disrupting antibody binding

• Antibody selection:

  • Choose antibodies specifically validated for IP applications (several are available according to search results)

  • Consider using antibodies targeting exposed epitopes for better accessibility

  • Determine optimal antibody-to-lysate ratio through titration experiments

• Pull-down strategy:

  • Pre-clear lysates to reduce non-specific binding

  • Use protein A/G beads appropriate for your antibody's species and isotype

  • Consider crosslinking antibodies to beads to prevent antibody co-elution

  • Extend incubation times (overnight at 4°C) to enhance weak interactions

• Elution and detection considerations:

  • Use specialized high-percentage gels for Western blot analysis of immunoprecipitated DNAH5

  • Consider non-reducing conditions if the antibody's epitope is sensitive to reducing agents

  • Use gradient gels to better resolve DNAH5 from potential interacting partners

How can DNAH5 antibodies be utilized in studying primary ciliary dyskinesia (PCD) disease mechanisms?

DNAH5 antibodies serve as valuable tools for investigating PCD pathophysiology:

• Diagnostic applications:

  • Immunofluorescence analysis of nasal or bronchial biopsies using DNAH5 antibodies can help diagnose PCD caused by DNAH5 mutations

  • Combined with TEM analysis, DNAH5 immunostaining can confirm outer dynein arm defects

• Genotype-phenotype correlation studies:

  • Different DNAH5 mutations (missense, nonsense, frameshift) may result in varying degrees of protein expression or mislocalization

  • DNAH5 antibodies enable visualization of these differences across patient samples with different mutation types

• Organoid modeling:

  • DNAH5 antibodies have been successfully used to characterize DNAH5-mutated PCD airway organoids

  • This approach allows comparison of ciliary structure and function between normal and PCD organoids

• Therapeutic development:

  • Screening potential therapeutics that may rescue DNAH5 expression or localization

  • Monitoring DNAH5 restoration in gene therapy approaches

What techniques can be combined with DNAH5 immunostaining for comprehensive ciliary function analysis?

Multimodal approaches provide more comprehensive insights into ciliary function:

• Structural analysis combinations:

  • Transmission electron microscopy (TEM) to directly visualize outer dynein arm defects

  • Super-resolution microscopy combined with DNAH5 immunostaining for nanoscale localization

  • Co-immunostaining with other ciliary markers (acetylated tubulin, other dynein components)

• Functional assessment techniques:

  • High-speed videomicroscopy to correlate DNAH5 localization with ciliary beat patterns

  • Ciliary beat frequency analysis alongside DNAH5 immunostaining

  • Mucociliary clearance assays to link DNAH5 defects with functional outcomes

• Molecular approaches:

  • Single-cell RNA sequencing (scRNA-Seq) to correlate DNAH5 protein expression with transcriptional profiles

  • Proteomics analysis to identify DNAH5 interaction partners and post-translational modifications

  • CRISPR-Cas9 genome editing to create DNAH5 mutations that mimic patient variants

• Disease modeling integration:

  • Patient-derived airway organoids combined with DNAH5 immunostaining

  • Air-liquid interface cultures to study DNAH5 in differentiated respiratory epithelium

  • Mouse models of DNAH5 mutations for in vivo correlation

How can DNAH5 antibodies be used to investigate the relationship between ciliary dysfunction and immune response?

Recent research has revealed connections between DNAH5 mutations and immune responses:

• Inflammation assessment:

  • Co-staining for DNAH5 and inflammatory markers in patient samples or organoids

  • Correlation of DNAH5 defects with expression of inflammatory cytokines

  • Analysis of immune cell recruitment in tissues with DNAH5 mutations

• Signaling pathway investigations:

  • Research has shown that DNAH5 mutations affect TGF-β/BMP and Notch signaling pathways, which regulate ciliated cell induction

  • These pathways also influence inflammatory cytokine expression

  • Combinatorial staining for DNAH5 and pathway components can reveal mechanistic connections

• Infection response studies:

  • Analysis of pathogen clearance in normal versus DNAH5-deficient respiratory epithelia

  • Investigation of immune receptor expression and activation in DNAH5-mutated cells

  • Assessment of antimicrobial peptide production in relation to DNAH5 status

• Therapeutic implications:

  • Evaluation of anti-inflammatory interventions on DNAH5-mutated tissues

  • Investigation of immune modulation as an adjunct therapy for PCD

  • Development of biomarkers for disease progression based on DNAH5 status and immune parameters

What are the considerations for using DNAH5 antibodies in single-cell imaging applications?

Single-cell analysis with DNAH5 antibodies requires specific technical considerations:

• Sample preparation optimization:

  • Gentle dissociation techniques to preserve ciliary structures

  • Fixation protocols that maintain cellular and subcellular architecture

  • Permeabilization methods that allow antibody access without disrupting delicate structures

• Advanced imaging approaches:

  • Super-resolution techniques (STED, STORM, PALM) to visualize DNAH5 distribution within individual cilia

  • Live-cell imaging using membrane-permeable antibody fragments or nanobodies

  • Correlative light and electron microscopy to link DNAH5 localization with ultrastructural features

• Quantitative analysis methods:

  • Automated image analysis workflows for unbiased quantification of DNAH5 signal intensity and distribution

  • Machine learning algorithms to classify ciliary phenotypes based on DNAH5 patterns

  • 3D reconstruction of DNAH5 distribution throughout the ciliary axoneme

• Combinatorial approaches:

  • Integration with single-cell transcriptomics or proteomics

  • Multiplexed immunofluorescence to simultaneously detect multiple ciliary components

  • Functional correlation with ciliary beat patterns at the single-cell level

How do different DNAH5 antibodies compare in detecting various mutant forms of DNAH5 protein?

Different DNAH5 mutations can affect antibody detection in various ways:

• Epitope accessibility considerations:

  • Antibodies targeting different regions of DNAH5 may exhibit varying abilities to detect mutant forms

  • Mutations may cause conformational changes that mask or expose different epitopes

  • Some mutations completely eliminate protein expression, while others result in truncated or mislocalized protein

• Mutation type effects:

  • Nonsense and frameshift mutations often result in complete loss of DNAH5 protein (null detection)

  • Missense mutations may preserve protein expression but alter localization or function

  • Splicing mutations can produce various truncated or aberrant proteins with unpredictable antibody reactivity

• Domain-specific antibody selection:

  • When studying specific DNAH5 mutations, select antibodies targeting domains either containing or distant from the mutation

  • For novel variants like c.12614G>A , compare results with multiple antibodies targeting different domains

• Validation approaches:

  • Use multiple antibodies targeting different DNAH5 epitopes

  • Compare antibody performance across samples with known DNAH5 mutations

  • Correlate antibody detection with genetic analysis to establish genotype-phenotype relationships

What methodological advances might improve DNAH5 protein detection in limited biological samples?

Working with limited patient samples requires optimized detection methods:

• Signal amplification techniques:

  • Tyramide signal amplification for immunofluorescence

  • Polymer-based detection systems for immunohistochemistry

  • Proximity ligation assays for detecting DNAH5 and its interaction partners

• Microfluidic approaches:

  • Microfluidic immunostaining to reduce antibody consumption

  • Single-cell Western blot techniques for analyzing DNAH5 in individual cells

  • Organ-on-chip platforms for functional studies with minimal sample requirements

• Alternative sample sources:

  • Nasal brush biopsies as less invasive alternatives to bronchoscopic sampling

  • Organoid derivation from minimal patient material for expanded testing

  • Conditional reprogramming of primary cells to generate sustainable cell lines

• Multiplexed detection strategies:

  • Cyclic immunofluorescence to detect multiple targets from single sections

  • Mass cytometry for simultaneous detection of DNAH5 and dozens of other proteins

  • Spatial transcriptomics combined with protein detection for correlative analysis

Data Table: DNAH5 Antibody Applications and Specifications