DNAL1 Antibody

What is DNAL1 and what are its primary biological functions?

DNAL1 (Dynein, Axonemal, Light Chain 1) is a protein component of the outer dynein arm complex in ciliary and flagellar structures. It serves as a critical regulatory element that links cytoskeletal components with the molecular motor dynein. Originally documented in Chlamydomonas as an outer arm dynein axonemal light chain, DNAL1 functions primarily as a scaffold for larger functional structures . Research has demonstrated that DNAL1 plays a crucial regulatory role for DNAH5 activity within the outer dynein arms of multiple motile structures including sperm flagella, respiratory cilia, and ependymal cilia .

The protein has significant clinical relevance as a potential candidate gene for primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic respiratory infections, abnormal left-right body asymmetry, and reduced fertility . Additionally, DNAL1 has been identified as an HIV dependency factor (HDF), suggesting potential importance as a therapeutic target in HIV treatment research .

DNAL1 has a calculated molecular weight of approximately 17 kDa (151 amino acids), though it typically appears at approximately 22 kDa in experimental detection systems . At least two distinct isoforms have been documented in the scientific literature .

How do I select the most appropriate DNAL1 antibody for my specific research application?

Selection of the optimal DNAL1 antibody requires careful consideration of multiple experimental factors:

Epitope targeting considerations:

• C-terminal targeting antibodies (e.g., ABIN6990951) recognize epitopes within the last 50 amino acids of human DNAL1

• N-terminal antibodies provide alternative detection options for different experimental contexts

• Some antibodies target specific amino acid regions (e.g., AA 41-190 or around Asn161)

Application compatibility matrix:

Species reactivity: When selecting antibodies, verify reactivity with your target species. While many DNAL1 antibodies react with human, mouse and rat samples, specific antibodies offer broader reactivity including cow, dog, rabbit, monkey and pig models .

To ensure optimal experimental outcomes, perform validation testing with appropriate positive controls (e.g., HeLa cells, brain tissue samples) which have demonstrated consistent DNAL1 expression across multiple studies .

What are the recommended protocols for Western blot detection of DNAL1?

Western blot detection of DNAL1 requires specific considerations due to its molecular characteristics:

Sample preparation:

• Extract total protein from tissue or cell lines expressing DNAL1 (brain tissue and HeLa cells have demonstrated consistent expression)

• Use RIPA or similar lysis buffer with protease inhibitors

• Quantify protein concentration using Bradford or BCA assay

Recommended western blot protocol:

• Load 20-30 μg of total protein per lane

• Separate proteins using 12-15% SDS-PAGE gels (optimal for lower molecular weight proteins)

• Transfer to PVDF or nitrocellulose membrane (PVDF may provide better retention of small proteins)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary DNAL1 antibody at dilutions between 1:1000-1:8000 in blocking buffer overnight at 4°C

• Wash 3x with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG) at 1:5000 for 1 hour

• Develop using ECL reagent and appropriate imaging system

Important considerations:

• Expected molecular weight is approximately 22 kDa, though calculated weight is 17 kDa

• Include positive controls such as HeLa cell lysate or brain tissue

• For reproducibility, maintain consistent antibody lots when possible

• Some tissues may require optimization of extraction methods to preserve DNAL1 integrity

How should I design experiments to investigate DNAL1's role in primary ciliary dyskinesia models?

Investigating DNAL1's role in primary ciliary dyskinesia (PCD) requires multifaceted experimental approaches:

Cellular model systems:

• Primary ciliated cell cultures: Isolated human nasal epithelial cells or bronchial epithelial cells cultured at air-liquid interface

• Immortalized respiratory cell lines: With induced ciliation

• Mouse models: Either DNAL1 knockout/knockdown or naturally occurring PCD models

Experimental design framework:

Critical methodological considerations:

• When performing immunofluorescence, optimize fixation methods (4% PFA typically works well)

• For co-IP experiments, use gentle lysis conditions to preserve protein-protein interactions

• When analyzing ciliary beating patterns, capture at 200+ frames per second and analyze multiple parameters (frequency, amplitude, coordination)

• Include both wild-type and known PCD mutation carriers as controls

• Validate findings across multiple cell types/donors to account for heterogeneity

This experimental framework enables systematic investigation of DNAL1's functional role in ciliary biology and its contribution to PCD pathogenesis.

Why do I observe differences between calculated and observed molecular weights when detecting DNAL1?

The discrepancy between DNAL1's calculated molecular weight (approximately 17 kDa) and its observed molecular weight in experimental systems (typically 22 kDa) represents a common phenomenon in protein research. Several factors contribute to this variation:

Biological factors affecting molecular weight discrepancies:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can significantly increase apparent molecular weight

• Protein isoforms: At least two distinct isoforms of DNAL1 have been documented , which may run at different molecular weights

• Structural characteristics: The three-dimensional conformation of DNAL1 may affect its migration pattern

• Protein-protein interactions: Some protein complexes may be resistant to denaturation

Technical considerations for accurate molecular weight assessment:

When troubleshooting molecular weight discrepancies:

• Include recombinant DNAL1 as a positive control when available

• Use molecular weight markers that are appropriate for low molecular weight proteins

• Consider running longer gels for better resolution in the 15-25 kDa range

• Document the specific antibody, conditions, and observed molecular weight in your experimental records to facilitate reproducibility

These methodological approaches help reconcile the differences between theoretical and observed molecular weights, improving experimental interpretation and reproducibility.

How can I improve signal specificity when using DNAL1 antibodies in immunohistochemistry or immunofluorescence?

Optimizing signal specificity for DNAL1 detection in tissue and cellular imaging requires attention to several critical parameters:

Fixation and permeabilization optimization:

• Test multiple fixation protocols (4% PFA, methanol, or combination approaches)

• Optimize permeabilization time and detergent concentration (0.1-0.5% Triton X-100 or 0.05-0.1% Saponin)

• Consider antigen retrieval methods for formalin-fixed tissue sections

Antibody validation strategies:

• Perform peptide competition assays with the immunizing peptide

• Include DNAL1 knockdown/knockout samples as negative controls

• Compare staining patterns across multiple antibodies targeting different DNAL1 epitopes

• Validate co-localization with established ciliary markers

Protocol optimization recommendations:

Advanced approaches for improved specificity:

• Use tyramide signal amplification for weak signals while maintaining specificity

• Consider super-resolution microscopy techniques for precise localization studies

• Implement automated image analysis with appropriate thresholding to distinguish signal from background

• For tissues with high autofluorescence, consider Sudan Black B treatment or spectral unmixing

These methodological considerations should significantly improve the specificity and reliability of DNAL1 detection in immunohistochemistry and immunofluorescence applications.

How can DNAL1 antibodies be utilized to investigate its role as an HIV dependency factor?

DNAL1's identification as an HIV dependency factor (HDF) opens important research directions for understanding viral pathogenesis. DNAL1 antibodies can be instrumental in elucidating these mechanisms through several experimental approaches:

Infection model systems for studying DNAL1-HIV interactions:

• Cell line models: CD4+ T cell lines, macrophage models, and transfected HeLa cells

• Primary cell systems: PBMCs, isolated CD4+ T cells from healthy donors

• Tissue explant models: Human lymphoid tissue explants

Methodological framework for investigating DNAL1 in HIV biology:

Advanced experimental considerations:

• Perform time-course experiments to determine when DNAL1 is most critical during the viral life cycle

• Use multiple viral strains (R5, X4, dual-tropic) to identify strain-specific dependencies

• Create domain-specific mutants of DNAL1 to map interaction sites with viral components

• Develop cell lines with tagged DNAL1 for live-cell imaging during infection

• Consider proteomics approaches (BioID, APEX) to identify the broader DNAL1 interactome during infection

This multifaceted approach leveraging DNAL1 antibodies can significantly advance understanding of how this cytoskeletal regulatory protein contributes to HIV replication and potential therapeutic targeting.

What approaches are recommended for studying DNAL1 post-translational modifications in ciliopathies?

Investigating DNAL1 post-translational modifications (PTMs) in ciliopathy contexts requires sophisticated methodological approaches:

Sample preparation considerations:

• Rapidly harvest and process samples to preserve labile PTMs

• Include phosphatase/deubiquitinase inhibitors in lysis buffers

• Consider enrichment strategies for ciliary/axonemal fractions

Comprehensive PTM analysis framework:

Advanced mass spectrometry approaches:

• Implement parallel reaction monitoring (PRM) for targeted PTM detection

• Use SILAC or TMT labeling to compare PTM profiles between normal and ciliopathic samples

• Apply enrichment strategies specific to phosphopeptides (TiO2, IMAC) or ubiquitinated peptides (K-ε-GG antibodies)

• Consider top-down proteomics for intact protein analysis to preserve PTM combinations

Functional validation methods:

• Generate site-specific mutants (phosphomimetic/phosphodeficient) to assess functional consequences

• Implement live-cell imaging with PTM-specific biosensors when applicable

• Correlate PTM changes with ciliary beating frequency and waveform analysis

• Assess interaction changes (using co-IP with DNAL1 antibodies) resulting from PTM alterations

These methodologies provide a comprehensive approach to characterizing DNAL1 post-translational modifications and their functional significance in ciliopathies, offering insights into potential therapeutic targeting strategies.

How should researchers interpret contradictory results obtained with different DNAL1 antibodies?

Contradictory results when using different DNAL1 antibodies present a common challenge that requires systematic troubleshooting and careful interpretation:

Sources of antibody-related result discrepancies:

• Epitope accessibility: Different antibodies target distinct regions of DNAL1 (C-terminus, N-terminus, central regions) , which may be differentially accessible in certain experimental contexts

• Isoform specificity: At least two DNAL1 isoforms exist , and antibodies may preferentially detect specific isoforms

• Cross-reactivity: Some antibodies may cross-react with related dynein light chain family members

• Application optimization: Antibodies optimized for one application (e.g., WB) may perform poorly in others (e.g., IF)

Systematic resolution approach:

Interpretation framework for contradictory results:

• When antibodies targeting different epitopes show discrepant results, consider protein conformation or complex formation affecting epitope accessibility

• If different molecular weights are detected, investigate potential isoforms or post-translational modifications

• When subcellular localization differs between antibodies, consider compartment-specific protein modifications or interactions

• For differences in expression levels, evaluate antibody affinity and optimal working concentrations

What are the best practices for quantitative analysis of DNAL1 expression in comparative studies?

Rigorous quantitative analysis of DNAL1 expression requires careful experimental design and appropriate statistical approaches:

Sample preparation considerations:

• Ensure equivalent amounts of starting material across comparison groups

• Process all samples simultaneously to minimize batch effects

• Include appropriate normalization controls (housekeeping proteins, total protein stains)

• Consider subcellular fractionation to enrich for ciliary/axonemal components

Quantification methodology framework:

Statistical analysis recommendations:

• Perform power analysis to determine appropriate sample sizes (minimum n=3 biological replicates)

• Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

• Account for multiple testing corrections when analyzing multiple conditions

• Consider mixed-effects models when dealing with repeated measures or nested experimental designs

• Report effect sizes alongside p-values for meaningful interpretation

Data visualization best practices:

These methodological approaches ensure robust quantitative analysis of DNAL1 expression across experimental conditions, facilitating reliable comparative studies and reproducible findings.