DNAAF3 Antibody

What is DNAAF3 and why is it important in scientific research?

DNAAF3, previously designated as C19orf51, is a 588 amino acid protein encoded by a gene located on human chromosome 19q13. It serves a critical function in ciliary biology as an essential factor for the preassembly of dynein motor complexes before their transport into cilia. DNAAF3 specifically facilitates the assembly of both axonemal inner and outer dynein arms, making it crucial for proper ciliary function. Research interest in DNAAF3 has intensified following the identification of loss-of-function mutations in patients with Primary Ciliary Dyskinesia (PCD), a rare autosomal-recessive disease characterized by recurrent respiratory infections and infertility . Studying DNAAF3 provides valuable insights into fundamental mechanisms of ciliary assembly and the pathogenesis of ciliopathies.

What types of DNAAF3 antibodies are available for research applications?

Several types of DNAAF3 antibodies are available for research, varying in their epitope specificity, host species, reactivity, and applications:

• Epitope specificity: Antibodies targeting different amino acid regions, including:

  • AA 1-209 (N-terminal region)

  • AA 151-250 (Central region)

  • AA 181-210 (Central region)

  • AA 50-200 (N-terminal region)

• Host and clonality: Most commercially available DNAAF3 antibodies are rabbit polyclonal antibodies .

• Species reactivity: Antibodies with specificity for human, mouse, or rat DNAAF3 are available, with some showing cross-reactivity between species .

• Applications: DNAAF3 antibodies are validated for various techniques including Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), ELISA, and Immunocytochemistry (ICC) .

• Conjugation options: Available as unconjugated or conjugated to various labels including HRP, FITC, biotin, and fluorescent dyes (e.g., AbBy Fluor® 594, 647, 750) .

How should I determine which DNAAF3 antibody epitope is most suitable for my research?

When selecting a DNAAF3 antibody epitope, consider your specific research objectives and experimental conditions:

• N-terminal epitopes (AA 1-209, AA 50-200):

  • Ideal for detecting full-length DNAAF3 protein

  • Useful in studies examining protein-protein interactions where the N-terminus plays a crucial role

  • May be less accessible in fixed tissues depending on protein conformation

  • Best suited for denatured applications like Western blotting

• Central region epitopes (AA 151-250, AA 181-210):

  • Often provide better accessibility in fixed tissues for immunohistochemistry

  • Useful for localization studies as these regions may be more exposed in the native protein

  • Demonstrated effectiveness in mouse samples for Western blotting and IHC applications

• Application-specific considerations:

  • For co-immunoprecipitation studies, choose antibodies that recognize native protein conformations

  • For immunofluorescence applications, validated antibodies like those used for HEK 293 cells at 4 μg/ml concentration after PFA fixation show reliable results

  • For cross-species studies, verify the sequence homology in your target epitope region

The optimal epitope choice may require empirical testing across multiple antibodies in your specific experimental system.

What are the optimized protocols for DNAAF3 immunofluorescence staining?

For optimal DNAAF3 immunofluorescence detection, follow these methodological guidelines:

• Sample preparation:

  • Fixation: 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature

  • Permeabilization: 0.3% Triton X-100 for 5-10 minutes

• Immunostaining protocol:

  • Blocking: 5-10% normal serum (matching secondary antibody species) for 1 hour

  • Primary antibody: Incubate with DNAAF3 antibody at 4 μg/ml (for HEK 293 cells) or at manufacturer-recommended dilution, overnight at 4°C

  • Secondary antibody: Species-appropriate fluorophore-conjugated antibody (1:500-1:1000) for 1 hour

  • Nuclear counterstain: DAPI (1:1000) for 5 minutes

  • Mounting: Anti-fade mounting medium to preserve fluorescence

• Special considerations:

  • Co-staining with ciliary markers (acetylated tubulin, ARL13B) can help establish spatial context

  • Z-stack imaging is recommended to capture the full three-dimensional ciliary structure

  • DNAAF3 typically shows cytoplasmic punctate staining consistent with its role in dynein preassembly rather than localization within cilia

  • For multiciliated cells, ensure imaging parameters capture the full range of expression levels

• Controls:

  • Positive control: Known DNAAF3-expressing ciliated tissues (respiratory epithelium)

  • Negative control: Secondary antibody-only staining to assess background

  • Specificity control: DNAAF3 knockdown or knockout samples when available

How can I validate the specificity of DNAAF3 antibodies in my experimental system?

Comprehensive validation of DNAAF3 antibodies requires multiple complementary approaches:

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Compare staining between control and DNAAF3-depleted samples

  • CRISPR/Cas9 knockout: Generate DNAAF3-null cells as definitive negative controls

  • Overexpression: Transfect cells with tagged DNAAF3 constructs and confirm co-localization with antibody staining

• Biochemical validation strategies:

  • Western blot: Confirm single band at expected molecular weight (~65 kDa)

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Immunoprecipitation-mass spectrometry: Verify DNAAF3 as the major precipitated protein

• Orthogonal validation methods:

  • Multiple antibodies: Compare staining patterns using antibodies targeting different DNAAF3 epitopes

  • Correlation with mRNA expression: Verify concordance between protein and transcript levels

  • Functional assays: Correlate antibody staining with known DNAAF3 biological activities

• Application-specific validation:

  • For immunofluorescence: Demonstrate expected subcellular localization pattern

  • For Western blotting: Show appropriate molecular weight and response to experimental manipulation

  • For immunoprecipitation: Verify enrichment of known DNAAF3 interaction partners

Document all validation experiments methodically to establish antibody reliability for your research applications.

What are the critical considerations for using DNAAF3 antibodies in Western blot analysis?

Successful Western blot analysis of DNAAF3 requires attention to several technical details:

• Sample preparation optimization:

  • Extraction buffer: RIPA buffer with protease inhibitors effectively solubilizes DNAAF3

  • Protein concentration: Load 20-50 μg total protein per lane

  • Denaturation: Complete denaturation (95°C for 5 minutes) is essential for accurate size determination

• Electrophoresis and transfer parameters:

  • Gel percentage: 8-10% SDS-PAGE gels provide optimal resolution for DNAAF3 (~65 kDa)

  • Transfer conditions: Standard semi-dry or wet transfer to PVDF membrane (preferred over nitrocellulose)

  • Transfer confirmation: Use reversible staining (Ponceau S) to verify efficient transfer

• Immunoblotting protocol:

  • Blocking: 5% non-fat dry milk in TBST for 1 hour

  • Primary antibody: Start with 1:1000 dilution (optimize based on specific antibody)

  • Incubation: Overnight at 4°C with gentle agitation

  • Secondary antibody: HRP-conjugated at 1:5000-1:10000 for 1 hour

  • Detection: Enhanced chemiluminescence (ECL) with appropriate exposure times

• Expected results and interpretation:

  • Full-length DNAAF3: Primary band at approximately 65 kDa

  • Multiple bands may indicate splice variants, post-translational modifications, or degradation products

  • Verify specificity using DNAAF3-depleted samples as negative controls

• Troubleshooting considerations:

  • Weak signal: Increase antibody concentration, extend incubation time, or use signal enhancement systems

  • High background: Increase blocking stringency, extend washing steps, or try alternative blocking agents

  • Multiple bands: Compare with literature reports and perform additional validation experiments

How can I use DNAAF3 antibodies to investigate dynein assembly defects in Primary Ciliary Dyskinesia (PCD)?

DNAAF3 antibodies provide valuable tools for investigating PCD-associated dynein assembly defects:

• Comparative localization analysis:

  • Compare DNAAF3 distribution patterns between healthy and PCD patient samples

  • Examine potential alterations in expression levels or subcellular localization

  • Assess whether DNAAF3 mutations affect protein stability or localization

• Dynein component colocalization studies:

  • Use DNAAF3 antibodies alongside antibodies against dynein components like DNAH5, DNAH9, DNAI2, and DNALI1

  • In DNAAF3-mutated PCD samples, expect absence or mislocalization of these components

  • Quantify colocalization coefficients between DNAAF3 and dynein assembly intermediates

• Biochemical interaction profiling:

  • Perform co-immunoprecipitation with DNAAF3 antibodies in control and PCD samples

  • Compare interaction profiles to identify missing or abnormal interactions in PCD cells

  • Correlate biochemical findings with structural and functional ciliary defects

• Rescue experiments with wild-type protein:

  • In DNAAF3-mutant cells, reintroduce wild-type DNAAF3

  • Use antibodies to verify restored localization and interactions

  • Correlate molecular findings with functional recovery of ciliary motility

• Developmental and tissue-specific analyses:

  • Compare DNAAF3 expression and localization across different ciliated tissues

  • Analyze developmental timing of defects in PCD models

  • Investigate tissue-specific consequences of DNAAF3 dysfunction

What methodological approaches can I use to study the temporal dynamics of DNAAF3 during ciliogenesis?

Studying DNAAF3 temporal dynamics requires carefully designed experimental approaches:

• Time-course immunofluorescence analysis:

  • Collect samples at defined intervals during ciliogenesis (e.g., 0h, 12h, 24h, 48h, 72h)

  • Perform dual staining for DNAAF3 and ciliary markers (acetylated tubulin, ARL13B)

  • Quantify changes in DNAAF3 levels, distribution patterns, and colocalization with assembly factors

  • Document the 5.62-fold increase in DNAAF3 expression during cilia formation reported in the literature

• Synchronized ciliogenesis models:

  • Primary airway epithelial cells in air-liquid interface culture

  • Serum starvation-induced ciliogenesis in immortalized cell lines

  • Differentiation of multiciliated cells from basal cell populations

• Protein expression and interaction dynamics:

  • Western blot analysis of DNAAF3 at different ciliogenesis time points

  • Sequential co-immunoprecipitation to track changing interaction partners

  • Pulse-chase experiments to analyze DNAAF3 protein stability during ciliogenesis

• Advanced imaging approaches:

  • Fluorescence recovery after photobleaching (FRAP) to measure DNAAF3 mobility

  • Photo-convertible fusion proteins to track DNAAF3 movement during assembly

  • Super-resolution microscopy to resolve assembly intermediates at nanoscale resolution

• Correlation with functional outcomes:

  • Relate DNAAF3 dynamics to measurable ciliary growth parameters

  • Track assembly of dynein components in relation to DNAAF3 expression patterns

  • Correlate molecular events with the emergence of ciliary motility

How can I optimize co-immunoprecipitation protocols using DNAAF3 antibodies?

For effective co-immunoprecipitation (Co-IP) experiments with DNAAF3 antibodies:

• Lysis buffer optimization:

  • Use gentle non-denaturing buffers (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40)

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Consider low ionic strength buffers to preserve weak interactions

• Pre-clearing and antibody binding:

  • Pre-clear lysate with Protein A/G beads (1 hour at 4°C) to reduce non-specific binding

  • Use 2-5 μg of DNAAF3 antibody per 500 μg of total protein

  • Allow sufficient binding time (overnight at 4°C with gentle rotation)

  • Consider cross-linking antibody to beads for cleaner results

• Washing and elution optimization:

  • Perform 4-5 washes with lysis buffer to remove non-specific binders

  • Consider including graduated salt washes to distinguish strong from weak interactions

  • Elute proteins by boiling in SDS sample buffer or use gentler elution with competing peptides

• Controls and validation:

  • Input control: Save 5-10% of pre-IP lysate to confirm target presence

  • IgG control: Parallel IP with non-specific IgG to identify non-specific binding

  • Antibody-only control: Process antibody without lysate to identify antibody bands

  • Reciprocal IP: When possible, confirm interactions by IP with antibodies against interaction partners

• Analysis considerations:

  • Immunoblot for DNAAF3 to confirm successful IP

  • Probe for expected interaction partners (other dynein assembly factors)

  • For unknown interactions, consider mass spectrometry analysis

  • Verify novel interactions with additional techniques (proximity ligation, FRET)

How do I interpret DNAAF3 staining patterns in different cell and tissue types?

Correct interpretation of DNAAF3 staining patterns requires understanding its biological context:

• Normal DNAAF3 distribution patterns:

  • Cytoplasmic punctate staining: Typical pattern reflecting DNAAF3's role in cytoplasmic dynein preassembly

  • Perinuclear enrichment: Often observed in actively ciliating cells

  • Absence from mature cilia: Expected as DNAAF3 functions in preassembly before transport into cilia

• Cell type-specific considerations:

  • Respiratory epithelium: Strong expression in ciliated cells, cytoplasmic pattern

  • Testicular tissue: Expression in developing sperm cells, critical for flagellar formation

  • Neural tissues: Variable expression in ependymal cells lining ventricles

  • Cell lines: May show diffuse staining depending on ciliation state

• Distinguishing specific from non-specific staining:

  • Nuclear staining: Generally indicates non-specific binding

  • Universal staining of all structures: Suggests high background or antibody specificity issues

  • Membrane-only staining: Potential cross-reactivity with other proteins

• Comparative analysis approaches:

  • Always compare with known DNAAF3 expression patterns from literature

  • Use multiple antibodies targeting different epitopes to confirm patterns

  • Correlate protein localization with mRNA expression data

  • Compare with knockout/knockdown controls when available

What are the common technical challenges when using DNAAF3 antibodies and how can I address them?

Researchers commonly encounter several technical challenges with DNAAF3 antibodies:

• Weak or absent signal in immunostaining:

  • Optimize fixation: Test different fixatives (PFA, methanol, acetone) and durations

  • Enhance antigen retrieval: Try heat-induced (citrate or EDTA buffer) or enzymatic methods

  • Increase antibody concentration: Perform titration experiments (1:50 to 1:1000)

  • Extend incubation time: Overnight at 4°C may improve signal strength

  • Use signal amplification: Consider tyramide signal amplification or polymer detection systems

• High background in immunofluorescence:

  • Improve blocking: Increase blocking agent concentration (5-10% serum)

  • Extend washing: More frequent and longer washing steps with gentle agitation

  • Reduce secondary antibody concentration: Dilute to 1:1000 or higher

  • Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

  • Include detergent (0.1% Tween-20) in washing buffers

• Multiple bands in Western blot:

  • Optimize sample preparation: Use fresh samples with complete protease inhibition

  • Adjust running conditions: Use gradient gels for better resolution

  • Perform peptide competition: Pre-incubate antibody with immunizing peptide

  • Compare with lysates from DNAAF3-depleted cells

  • Consult literature for documented splice variants or post-translational modifications

• Inconsistent results between experiments:

  • Standardize protocols: Document all parameters and maintain consistency

  • Use internal controls: Include positive and negative controls in each experiment

  • Check antibody storage: Aliquot antibodies to avoid freeze-thaw cycles

  • Verify sample quality: Ensure consistent sample collection and processing

  • Consider lot-to-lot variability: Test new antibody lots against previous results

How do I analyze DNAAF3 expression in ciliopathy models and patient samples?

Analyzing DNAAF3 in ciliopathy models requires careful experimental design and interpretation:

• Patient sample analysis considerations:

  • Match controls carefully by age, sex, and tissue type

  • Consider disease heterogeneity when interpreting results

  • Use multiple methodologies (protein and mRNA analysis)

  • Compare findings with established disease markers

• Quantitative analysis approaches:

  • Western blot densitometry: Normalize DNAAF3 levels to appropriate housekeeping proteins

  • Quantitative immunofluorescence: Measure intensity relative to defined cellular landmarks

  • qRT-PCR: Correlate protein findings with transcript levels

  • Image analysis: Use automated tools for unbiased quantification of localization patterns

• Functional correlation strategies:

  • Ciliary ultrastructure: Correlate DNAAF3 findings with TEM analysis of axonemal structures

  • Ciliary motility: Link molecular observations to functional ciliary beating

  • Developmental phenotypes: Connect DNAAF3 alterations to organismal manifestations

  • Dynein assembly: Assess impact on other components (DNAH5, DNAH9, DNAI2, DNALI1)

• Model system comparisons:

  • In vitro models: Cell lines with DNAAF3 mutations or knockdown

  • Ex vivo models: Primary cultures from patient samples

  • Animal models: DNAAF3-mutant mice or other model organisms

  • iPSC-derived systems: Patient-specific differentiated ciliated cells

• Genetic background considerations:

  • Document specific DNAAF3 mutations in your samples

  • Consider potential modifier genes in the interpretation of phenotypic variability

  • Compare with known mutation databases for PCD and other ciliopathies

How can DNAAF3 antibodies contribute to understanding the molecular pathogenesis of ciliopathies?

DNAAF3 antibodies offer valuable tools for elucidating ciliopathy pathogenesis:

• Molecular diagnosis applications:

  • Develop diagnostic immunostaining panels for PCD screening

  • Establish DNAAF3 protein expression patterns as biomarkers for specific PCD subtypes

  • Correlate DNAAF3 protein alterations with specific genetic mutations and clinical phenotypes

• Pathogenic mechanism investigations:

  • Map the sequence of molecular events in dynein assembly defects

  • Identify which assembly steps are disrupted in different DNAAF3 mutations

  • Determine whether DNAAF3 dysfunction affects specific dynein subtypes selectively

  • Investigate potential compensatory mechanisms in different tissues

• Therapeutic development applications:

  • Screen for compounds that stabilize mutant DNAAF3 protein

  • Test gene therapy approaches using DNAAF3 antibodies to verify protein expression

  • Evaluate small molecule chaperones that might restore assembly function

  • Assess phenotypic rescue in cellular and animal models

• Systems biology approaches:

  • Integrate DNAAF3 into comprehensive models of ciliogenesis

  • Map the complete DNAAF3 interactome in healthy and disease states

  • Identify regulatory networks controlling DNAAF3 expression and function

  • Compare DNAAF3-related pathways across different ciliopathies

• Evolutionary and comparative studies:

  • Examine DNAAF3 conservation and divergence across species

  • Compare ciliary assembly mechanisms between motile and primary cilia

  • Investigate tissue-specific adaptations in DNAAF3 function

What emerging technologies might enhance DNAAF3 antibody applications in ciliary research?

Several emerging technologies promise to expand DNAAF3 antibody applications:

• Advanced imaging technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale localization

  • Expansion microscopy to physically enlarge ciliary structures

  • Lattice light-sheet microscopy for dynamic studies of DNAAF3 in living cells

  • Cryo-electron tomography combined with immunolabeling for structural context

• Proximity labeling approaches:

  • BioID fusion proteins to identify proteins in proximity to DNAAF3

  • APEX2 for electron microscopy-compatible proximity labeling

  • Split-BioID for studying conditional or transient interactions

  • Multiplexed proximity labeling to map assembly complexes

• Single-cell analysis methods:

  • Single-cell proteomics to analyze DNAAF3 expression heterogeneity

  • Spatial transcriptomics combined with protein detection

  • Mass cytometry (CyTOF) with DNAAF3 antibodies for high-parameter analysis

  • Microfluidic approaches for ciliary protein dynamics in single cells

• Protein engineering applications:

  • Nanobodies against DNAAF3 for improved tissue penetration

  • Optogenetic tools to manipulate DNAAF3 localization and function

  • Degron-based systems for rapid DNAAF3 depletion

  • CRISPR-based endogenous tagging for physiological protein tracking

• Computational approaches:

  • Machine learning for automated analysis of ciliary phenotypes

  • Molecular dynamics simulations of DNAAF3-dynein interactions

  • Systems biology modeling of assembly pathways

  • Predictive algorithms for antibody epitope accessibility