DNAI1 Antibody

What is DNAI1 and what is its biological significance in research?

DNAI1 encodes the protein 'dynein axonemal intermediate chain 1' in humans, which functions as a critical component of the outer dynein arm complex in ciliary and flagellar structures. This protein may also be known by alternative names including Dnaic1, CILD1, DIC1, ICS1, PCD, dynein intermediate chain 1 (axonemal), and dynein axonemal intermediate polypeptide 1. Structurally, the protein has a reported molecular mass of approximately 79.3 kilodaltons, though observed molecular weights in experimental systems typically range between 80-85 kDa .

DNAI1 has significant research relevance due to its role in ciliary motility and its association with primary ciliary dyskinesia (PCD), a genetic disorder characterized by impaired ciliary function. The protein is predominantly expressed in tissues containing motile cilia, including respiratory epithelium and the male reproductive tract, making DNAI1 antibodies valuable tools for studying ciliopathies and ciliary dynamics.

What are the primary experimental applications for DNAI1 antibodies?

Based on validated research applications, DNAI1 antibodies are used in multiple experimental contexts:

These applications enable researchers to investigate DNAI1 expression, localization, and function across various experimental systems relevant to ciliary biology and pathology .

What species reactivity should researchers consider when selecting DNAI1 antibodies?

When selecting DNAI1 antibodies, researchers should carefully evaluate species reactivity profiles. Many commercially available antibodies demonstrate cross-reactivity with multiple species:

It is essential to validate species reactivity experimentally even when manufacturers claim cross-reactivity. Sequence conservation in the epitope region is a critical determinant of cross-species reactivity .

How should researchers optimize Western blot protocols for DNAI1 detection?

Optimizing Western blot protocols for DNAI1 detection requires attention to several technical parameters:

Sample preparation is critical - DNAI1 is most abundantly expressed in ciliated tissues such as human brain tissue, mouse testis tissue, and rat testis tissue, which serve as excellent positive controls . When extracting protein, use lysis buffers containing protease inhibitors to prevent degradation of this 79.3 kDa protein.

For optimal detection, the recommended antibody dilution range is 1:500-1:2000 for Western blotting applications . Due to the observed molecular weight range (80-85 kDa), use 8-10% acrylamide gels for optimal resolution in this molecular weight range. Extended transfer times (90-120 minutes) may be necessary for complete transfer of this relatively large protein.

When interpreting results, note that DNAI1 typically appears as a band between 80-85 kDa, slightly higher than the calculated molecular weight of 79 kDa, which may reflect post-translational modifications .

What are the optimal protocols for immunofluorescence detection of DNAI1 in ciliated cells?

For effective immunofluorescence detection of DNAI1 in ciliated structures, researchers should implement the following optimization strategies:

Sample preparation is crucial - fixation with 4% paraformaldehyde is generally effective at preserving ciliary structures while maintaining epitope accessibility. For permeabilization, use mild detergents (0.1-0.2% Triton X-100) to preserve delicate ciliary architecture.

Antibody incubation requires careful optimization - the recommended dilution range for immunofluorescence applications is 1:400-1:1600 . Extended primary antibody incubation (overnight at 4°C) often improves signal-to-noise ratio for axonemal proteins like DNAI1.

For visualization, confocal microscopy with z-stack acquisition is preferred to capture the three-dimensional ciliary structures. Co-staining with markers such as acetylated α-tubulin helps confirm axonemal localization of DNAI1 signal. hTERT-RPE1 cells have been validated as useful positive controls for DNAI1 immunofluorescence .

How can researchers validate DNAI1 antibody specificity for critical experiments?

Thorough validation of DNAI1 antibody specificity is essential for generating reliable experimental data, particularly in disease-related studies. A comprehensive validation approach should include:

Genetic validation approaches:

• Use of DNAI1 knockout/knockdown models as negative controls

• Rescue experiments with tagged DNAI1 constructs

• Comparison with CRISPR-edited cell lines lacking DNAI1

Biochemical validation approaches:

• Peptide competition assays to confirm epitope specificity

• Western blot analysis comparing samples with differential DNAI1 expression

• Detection of recombinant DNAI1 protein as a positive control

Comparative analysis:

• Testing multiple antibodies targeting different DNAI1 epitopes

• Cross-validation using multiple detection methods (WB, IF, ELISA)

• Correlation with mRNA expression data from RT-PCR or RNA-seq

Publication citation records can provide evidence of antibody validation in peer-reviewed research contexts, with several antibodies showing validation in published studies examining ciliary dysfunction .

How should researchers approach DNAI1 antibody use in primary ciliary dyskinesia (PCD) studies?

When investigating PCD using DNAI1 antibodies, researchers should implement specialized experimental designs:

PCD represents a clinically and genetically heterogeneous disorder, with DNAI1 mutations accounting for approximately 10% of cases. Different mutations may have varying effects on protein expression, localization, and function. Some mutations result in complete absence of the protein, while others produce non-functional or mislocalized protein.

For experimental design, include appropriate controls: samples from PCD patients with known DNAI1 mutations serve as valuable disease controls, while matched samples from healthy individuals provide essential normal controls. When available, include non-PCD ciliopathy samples to demonstrate specificity of DNAI1-related findings.

What approaches are recommended for studying DNAI1 protein interactions with other axonemal components?

Investigating DNAI1 protein interactions requires specialized immunoprecipitation protocols and complementary techniques:

Co-immunoprecipitation optimization:

• Use ciliated cell types with high DNAI1 expression (e.g., human bronchial epithelial cells)

• Employ gentle lysis conditions (150-300 mM NaCl, 0.5-1% non-ionic detergents)

• Include protease inhibitors to preserve protein complexes

• Consider crosslinking approaches for stabilizing transient interactions

Interaction validation methods:

• Western blot detection of known DNAI1 interactors (DNAH5, DNAI2, etc.)

• Mass spectrometry for unbiased identification of novel interaction partners

• Proximity ligation assay (PLA) for confirming interactions in situ

• Reciprocal co-IP experiments to verify specific interactions

For interpreting interaction data, consider the hierarchical assembly of dynein complexes and the potential for both direct and indirect interactions within the multiprotein axonemal structure.

How do post-translational modifications affect DNAI1 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of DNAI1, requiring special experimental considerations:

The discrepancy between calculated (79 kDa) and observed (80-85 kDa) molecular weights suggests the presence of PTMs on DNAI1 . These modifications may include phosphorylation, which can regulate dynein assembly and function, and ubiquitination, which may control protein turnover.

PTMs can affect antibody detection through epitope masking (physically blocking antibody binding sites), inducing conformational changes (altering epitope accessibility), or causing electrophoretic mobility shifts (changing apparent molecular weight).

To address PTM influences, researchers should:

• Use multiple antibodies targeting different DNAI1 epitopes

• Consider phosphatase or deubiquitinase treatment before detection

• Apply PTM-specific enrichment strategies for comprehensive analysis

• Employ mass spectrometry to identify and map specific modification sites

How should researchers troubleshoot inconsistent DNAI1 antibody performance?

When encountering variability in DNAI1 antibody performance, systematic troubleshooting approaches are essential:

Sample preparation factors:

• Fixation conditions: Overfixation may mask epitopes; reduce fixation times if needed

• Buffer composition: Optimize pH and ionic strength to improve epitope accessibility

• Sample storage: Minimize protein degradation through proper storage protocols

Antibody-specific considerations:

• Evaluate lot-to-lot variability by comparing performance between antibody lots

• Prevent antibody degradation by avoiding repeated freeze-thaw cycles

• Address non-specific binding by optimizing blocking conditions and washing steps

Detection system optimization:

• Employ signal amplification techniques for detecting low-abundance DNAI1

• Test alternative secondary antibodies or detection systems

• Calibrate imaging parameters for consistent detection sensitivity

Proteintech's DNAI1 antibody (12756-1-AP) has been validated in multiple applications and species, making it a useful benchmark for comparison when troubleshooting other antibodies .

What are the recommended positive and negative controls for DNAI1 antibody validation?

Appropriate controls are critical for validating DNAI1 antibody specificity and performance:

Recommended positive controls:

• Human, mouse, and rat testis tissue (high DNAI1 expression)

• Human brain tissue (validated positive tissue)

• hTERT-RPE1 cells (validated for immunofluorescence)

• Respiratory epithelial cells with mature cilia

Effective negative controls:

• Samples from PCD patients with confirmed DNAI1 null mutations

• CRISPR-edited cell lines with DNAI1 knockout

• siRNA/shRNA DNAI1 knockdown samples

• Non-ciliated cell lines that do not express DNAI1

Using these carefully selected controls helps distinguish specific signal from background and validates antibody performance across different experimental systems.

How can researchers quantitatively assess DNAI1 expression in comparative studies?

For rigorous quantitative analysis of DNAI1 expression between experimental groups:

Western blot quantification approaches:

• Use appropriate loading controls (β-actin, GAPDH for whole-cell lysates; axonemal markers for ciliary preparations)

• Employ digital imaging systems with linear detection range

• Include calibration standards for absolute quantification

• Analyze multiple biological replicates (minimum n=3) for statistical validity

Immunofluorescence quantification methods:

• Standardize all acquisition parameters across experimental groups

• Perform z-stack imaging to capture complete ciliary structures

• Develop consistent regions of interest (ROIs) for signal intensity measurement

• Normalize DNAI1 signal to reference ciliary markers

Statistical analysis considerations:

• Apply appropriate statistical tests based on data distribution

• Consider paired analyses for matched samples

• Implement normalization strategies to account for sample variability

• Report effect sizes alongside statistical significance

These quantitative approaches enable robust comparative analysis of DNAI1 expression in normal versus pathological conditions or across experimental manipulations.

How are DNAI1 antibodies utilized in studying ciliopathies beyond PCD?

While DNAI1 mutations are primarily associated with PCD, DNAI1 antibodies have broader applications in ciliopathy research:

DNAI1 antibodies serve as valuable tools for assessing ciliary structure in various ciliopathies, even those not directly caused by DNAI1 mutations. By examining DNAI1 localization and abundance, researchers can evaluate secondary effects on axonemal organization in disorders affecting ciliary assembly, maintenance, or function.

In heterotaxy and other laterality disorders, DNAI1 antibodies help assess nodal cilia function during embryonic development. For male infertility investigations, these antibodies enable evaluation of sperm flagellar structure and dynein arm integrity.

DNAI1 antibodies also facilitate pharmacological studies testing compounds that may rescue ciliary function in various ciliopathies, providing readouts of treatment efficacy at the protein level.

What specialized techniques can enhance DNAI1 detection in challenging samples?

For difficult samples or applications requiring enhanced sensitivity:

Signal amplification strategies:

• Tyramide signal amplification (TSA) can significantly increase detection sensitivity

• Quantum dot-conjugated secondary antibodies provide improved signal stability

• Multiplex immunofluorescence with spectral unmixing for complex co-localization studies

Sample preparation enhancements:

• Antigen retrieval methods (heat-induced or enzymatic) for formalin-fixed tissues

• Specialized fixation protocols for preserving delicate ciliary structures

• Detergent optimization for improved epitope accessibility without structural damage

Advanced imaging approaches:

• Super-resolution microscopy (STED, STORM, SIM) for detailed localization studies

• Expansion microscopy for physically enlarging ciliary structures

• Live-cell imaging with tagged DNAI1 constructs to complement antibody studies

These specialized approaches enable DNAI1 detection in challenging samples such as archived clinical specimens, embryonic tissues, or samples with low ciliary abundance.