DNAH12 Antibody

What is DNAH12 and what are its key structural characteristics?

DNAH12 is a member of the dynein heavy chain protein family involved in axonemal structure formation. In humans, the canonical protein consists of 3092 amino acid residues with a molecular mass of 356.9 kDa and is primarily localized in the cytoplasm. Up to four different isoforms have been reported for this protein, which is notably expressed in the fallopian tube. DNAH12 functions as a marker for Bronchus Ciliated Cells and has several synonyms including axonemal dynein heavy chain isotype3, ciliary dynein heavy chain 12, dynein heavy chain domain-containing protein 2, and axonemal beta dynein heavy chain 12. Orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

What types of DNAH12 antibodies are currently available for research applications?

Multiple types of DNAH12 antibodies are available for research applications, including:

Researchers can select from affinity-isolated antibodies produced in rabbits and rats, with various applications including Western blot, immunohistochemistry, ELISA, and immunofluorescence techniques .

How is DNAH12 expression distributed across different tissues?

DNAH12 shows tissue-specific expression patterns that are important to consider when designing experiments. Based on RT-PCR, qPCR, and immunoblotting detection studies, DNAH12 mRNA and protein demonstrate high expression in testes with relatively lower expression in lungs, tracheas of male mice, and oviducts of female mice. Interestingly, in epididymides, DNAH12 protein is detected without corresponding mRNA expression, suggesting that the protein detected in epididymides originates from testes, likely from sperm cells. Developmentally, DNAH12 protein is initially detected in testes around 21 days postpartum (dpp) and maintains high expression levels thereafter .

What are the recommended protocols for using DNAH12 antibodies in immunohistochemistry?

For immunohistochemistry applications with DNAH12 antibodies, the following methodological approach is recommended:

• Tissue fixation: Use 4% paraformaldehyde fixation for optimal epitope preservation

• Dilution range: Use a dilution range of 1:500-1:1000 for primary antibody incubation

• Incubation conditions: Incubate at 4°C overnight with gentle rocking

• Antigen retrieval: Heat-mediated antigen retrieval using citrate buffer (pH 6.0) is recommended

• Blocking: Use 5% normal serum from the same species as the secondary antibody

• Secondary antibody: Select appropriate HRP-conjugated secondary antibody at 1:200-1:500 dilution

• Detection system: DAB (3,3'-diaminobenzidine) detection is commonly used

• Controls: Include both positive controls (tissues known to express DNAH12, such as testes) and negative controls (tissues without DNAH12 expression or primary antibody omission)

How can researchers validate the specificity of DNAH12 antibodies?

Validating antibody specificity is crucial for research reliability. For DNAH12 antibodies, a multi-method validation approach is recommended:

• Genetic validation: Compare antibody reactivity between wild-type (Dnah12+/+) and knockout (Dnah12-/-) mice tissues/cells

• Immunogen analysis: Verify the immunogen sequence uniqueness through bioinformatic analysis

• Pre-absorption testing: Pre-incubate the antibody with the immunogen peptide before staining

• Multiple antibody concordance: Compare results from different antibodies targeting distinct DNAH12 epitopes

• Orthogonal validation: Correlate protein detection with mRNA expression data

• Western blot analysis: Confirm single band of appropriate molecular weight (approximately 357 kDa)

• Immunoprecipitation followed by mass spectrometry: Confirm pull-down of DNAH12 and known interacting partners

Studies have employed these methods to validate DNAH12 antibodies, such as the polyclonal antibody generated against the 1-200 amino acid sequence of mouse DNAH12, which was verified using Dnah12+/+ and Dnah12-/- mice .

What are the optimal protein extraction methods for DNAH12 Western blot analysis?

DNAH12 is a large protein (356.9 kDa) that requires specific extraction and handling protocols:

• Tissue preparation: Flash-freeze tissue samples in liquid nitrogen before extraction

• Extraction buffer: Use specialized buffers containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Homogenization: Employ mechanical disruption with brief sonication pulses

• Low-speed centrifugation: Remove cellular debris (1,000g for 10 minutes)

• High-speed centrifugation: Clarify extract (20,000g for 30 minutes)

• Protein concentration: Use methods compatible with detergents (Bradford or BCA)

• Sample preparation: Add sample buffer with reducing agent and heat at 70°C (not boiling)

• Gel selection: Use low percentage (3-8%) gradient gels for proper resolution

• Extended transfer time: Employ overnight wet transfer at low voltage for complete transfer

• Detection: Use highly sensitive chemiluminescence systems for optimal visualization

How can DNAH12 antibodies be utilized to investigate male infertility mechanisms?

DNAH12 antibodies can serve as powerful tools in male infertility research through several methodological approaches:

• Immunohistochemical profiling of testicular biopsies:

  • Compare DNAH12 localization in fertile versus infertile men

  • Correlate with sperm parameters and fertilization outcomes

• Co-immunoprecipitation studies:

  • Investigate DNAH12 interactions with DNAH1 and DNALI1

  • Identify novel binding partners in the inner dynein arm complex

• Ultrastructural localization:

  • Perform immunogold electron microscopy to precisely localize DNAH12 within sperm flagella

  • Correlate with axonemal defects in infertile patients

• Mutation-specific antibodies:

  • Develop antibodies recognizing specific DNAH12 mutations

  • Study conformational changes in mutant proteins

• Quantitative analysis:

  • Perform Western blot and ELISA to quantify DNAH12 levels in sperm from patients with different fertility phenotypes

  • Correlate protein levels with specific mutations and flagellar defects

Recent research has demonstrated that bi-allelic mutations in DNAH12 are associated with asthenoteratozoospermia and male infertility, with DNAH12 playing a critical role in recruiting inner dynein arm components like DNAH1 and DNALI1 specifically in flagella but not in cilia .

What are the recommended approaches for studying DNAH12's role in axonemal organization?

To investigate DNAH12's role in axonemal organization, researchers should consider:

• Super-resolution microscopy:

  • Employ techniques like STORM or STED microscopy

  • Use dual-labeling with DNAH12 antibodies and other axonemal markers

  • Analyze co-localization patterns at nanometer resolution

• Protein-protein interaction mapping:

  • Perform proximity ligation assays to visualize DNAH12 interactions in situ

  • Use BioID or APEX2 proximity labeling with DNAH12 as bait

  • Validate interactions with co-immunoprecipitation and mass spectrometry

• Functional rescue experiments:

  • Express wild-type or mutant DNAH12 in knockout models

  • Assess flagellar structure and motility recovery

  • Use antibodies to confirm proper localization of rescue constructs

• Temporal expression analysis:

  • Track DNAH12 expression during spermatogenesis and ciliogenesis

  • Correlate with appearance of other axonemal components

  • Establish hierarchical assembly patterns

• Comparative analysis between cilia and flagella:

  • Use immunofluorescence to compare DNAH12 distribution between respiratory cilia and sperm flagella

  • Correlate with functional differences in axonemal organization

Research has shown that DNAH12 deficiency leads to axonemal defects characterized by impaired inner dynein arms and central pair loss in sperm flagella, while no such defects are observed in cilia, suggesting tissue-specific functions .

How can multiple antibody-based techniques be integrated to comprehensively study DNAH12 function?

An integrated multi-technique approach provides the most comprehensive understanding of DNAH12 function:

This multi-modal approach has been successfully employed to demonstrate that DNAH12 interacts with other inner dynein arm components like DNALI1 and DNAH1 in mouse testes, and disruption of DNAH12 impairs the recruitment of dynein components into sperm flagella .

What are common pitfalls in DNAH12 antibody experiments and how can they be addressed?

Researchers working with DNAH12 antibodies may encounter several technical challenges:

• High molecular weight detection issues:

  • Problem: Incomplete transfer of large proteins (356.9 kDa)

  • Solution: Use specialized transfer conditions with extended time, lower voltage, and addition of SDS to transfer buffer

• Cross-reactivity concerns:

  • Problem: Antibody cross-reactivity with related dynein proteins

  • Solution: Perform thorough validation with knockout controls and pre-absorption tests

• Tissue fixation artifacts:

  • Problem: Epitope masking during fixation

  • Solution: Optimize fixation time and test multiple antigen retrieval methods

• Isoform specificity:

  • Problem: Differential detection of the four DNAH12 isoforms

  • Solution: Select antibodies targeting common regions or use isoform-specific antibodies

• Background in immunohistochemistry:

  • Problem: High background in ciliated tissues

  • Solution: Use tyramide signal amplification for specific detection and stringent blocking protocols

• Reproducibility issues:

  • Problem: Lot-to-lot variation in antibody performance

  • Solution: Validate each new lot against previous standards and maintain positive control samples

Addressing these issues requires careful experimental design and appropriate controls, particularly genetic validation using Dnah12+/+ and Dnah12-/- tissues as demonstrated in recent studies .

What considerations should be made when selecting DNAH12 antibodies for specific research questions?

Selecting the appropriate DNAH12 antibody requires careful consideration of:

• Epitope location:

  • N-terminal (aa 1-200) antibodies: Best for detecting full-length protein

  • Middle region antibodies: Less affected by terminal modifications

  • C-terminal antibodies: May detect specific isoforms or processed forms

• Species cross-reactivity:

  • Human-specific antibodies: For clinical samples and human cell lines

  • Multi-species reactive antibodies: For comparative studies between models

  • Species-specific antibodies: For avoiding cross-reactivity in co-culture systems

• Application compatibility:

  • Conformation-sensitive antibodies: Better for immunoprecipitation and flow cytometry

  • Denaturation-resistant epitopes: Preferred for Western blot and IHC on fixed tissues

  • Native-state recognition: Essential for functional blocking studies

• Validation standards:

  • Genetic validation (knockout controls)

  • Orthogonal validation (correlation with mRNA)

  • Independent antibody validation (multiple antibodies to different epitopes)

  • Tissue expression profile validation

Commercial suppliers offer various DNAH12 antibodies with applications including Western blot, ELISA, immunofluorescence, and immunohistochemistry, with varying species reactivity profiles including human, rabbit, rat, dog, and horse .

How might DNAH12 antibodies contribute to emerging personalized medicine approaches for male infertility?

DNAH12 antibodies hold significant potential for advancing personalized medicine in male infertility:

• Diagnostic applications:

  • Development of immunodiagnostic assays to detect DNAH12 in sperm samples

  • Correlation of DNAH12 protein levels with specific genetic variants

  • Generation of mutation-specific antibodies for targeted diagnostics

• Patient stratification:

  • Classification of patients based on DNAH12 expression patterns

  • Prediction of treatment response based on molecular phenotyping

  • Identification of patient subgroups with specific axonemal defects

• Therapeutic monitoring:

  • Assessment of therapeutic interventions targeting dynein assembly

  • Monitoring of DNAH12 expression during treatment

  • Validation of gene therapy approaches for DNAH12 mutations

• Biomarker development:

  • Integration of DNAH12 detection into multi-marker panels

  • Correlation with clinical outcomes and fertility potential

  • Development of non-invasive detection methods

Recent research has identified bi-allelic mutations in DNAH12 in infertile males from six unrelated families, highlighting the potential clinical significance of DNAH12 testing in patients with asthenoteratozoospermia .

What novel methodological approaches might enhance DNAH12 antibody applications in developmental biology?

Emerging methodologies offer new opportunities for DNAH12 research:

• Organoid models:

  • Application of DNAH12 antibodies in testicular and respiratory organoids

  • Live-cell imaging using cell-permeable fluorescently-tagged DNAH12 antibodies

  • Analysis of DNAH12 dynamics during organoid development

• CRISPR-based approaches:

  • Endogenous tagging of DNAH12 for live visualization

  • Antibody validation using CRISPR knockout controls

  • Correlation of antibody signals with CRISPR-mediated mutations

• Single-cell technologies:

  • Integration of DNAH12 antibodies in single-cell proteomics

  • Correlation with single-cell transcriptomics data

  • Spatial profiling of DNAH12 in tissue sections

• In vivo imaging:

  • Development of near-infrared DNAH12 antibodies for in vivo imaging

  • Monitoring of DNAH12 expression during development

  • Assessment of therapeutic interventions targeting DNAH12

• High-throughput screening:

  • Antibody-based screens for compounds affecting DNAH12 expression or localization

  • Development of reporter systems for DNAH12 function

These methodologies build upon foundational research showing that DNAH12 expression begins around 21 days postpartum in mice, coinciding with key developmental stages in spermatogenesis .