DNAH7 Antibody, HRP conjugated

What is DNAH7 and why is it important in scientific research?

DNAH7 (Dynein Axonemal Heavy Chain 7) is a force-generating protein that plays a crucial role in respiratory cilia and sperm flagella beating. It functions by producing force toward the minus ends of microtubules and possesses ATPase activity, with the force-producing power stroke occurring upon ADP release . As a component of the inner dynein arm (IDA) of ciliary axonemes, DNAH7 is specifically found in IDAb and IDAe complexes and associates with the light intermediate chain DNALI1 .

Research importance:

• DNAH7 is associated with primary ciliary dyskinesia and male fertility outcomes

• Recent studies have identified DNAH7 mutations as potentially beneficial for colorectal cancer patients receiving immune checkpoint inhibitors

• It serves as a critical marker for studying ciliary function in respiratory epithelium

What applications are most appropriate for DNAH7 antibody, HRP conjugated?

DNAH7 antibody, HRP conjugated, is particularly suitable for:

• ELISA applications: The most validated application at dilutions of 1:500-1000

• Western blotting: For detection of DNAH7 protein (~461 kDa)

• Immunodetection following protein separation: Especially useful for quantitative analyses of DNAH7 expression levels in different tissue samples

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and reducing background. This makes it particularly valuable for high-throughput screening applications and situations requiring enhanced sensitivity .

What is the specificity profile of DNAH7 antibody and how has it been validated?

The specificity of DNAH7 antibody has been validated through multiple approaches:

• Western blot analysis: Detects a specific band at the expected size of DNAH7 (~461 kDa) in control samples

• Immunofluorescence microscopy: Shows localization throughout the entire length of ciliary axonemes in healthy control subjects

• Comparative studies: Shows absence of signal in respiratory ciliary axonemes from individuals with CCDC39 and CCDC40 variants, confirming specificity

Cross-reactivity testing has shown predicted reactivity with DNAH7 from multiple species including human, mouse, rat, dog, cow, sheep, pig, horse, chicken, and rabbit , though experimental validation has primarily been performed in human samples.

What is the recommended protocol for using DNAH7 antibody, HRP conjugated in ELISA?

Standard ELISA protocol for HRP-conjugated DNAH7 antibody:

• Coating: Coat wells with target protein/sample (50-100 μL/well)

• Blocking: Block with 5% non-fat milk or BSA in PBS-T (1-2 hours at room temperature)

• Primary antibody: Apply HRP-conjugated DNAH7 antibody at 1:500-1000 dilution in blocking buffer

• Incubation: 1-2 hours at room temperature or overnight at 4°C

• Washing: Wash 3-5 times with PBS-T

• Detection: Add TMB or other HRP substrate and measure colorimetric signal

• Analysis: Analyze results against appropriate controls

Important considerations:

• Optimal antibody concentration may require titration for your specific application

• Always include negative controls (no primary antibody) and positive controls (known DNAH7-expressing samples)

• Maintain consistent temperature conditions throughout the protocol

How should DNAH7 antibody, HRP conjugated be stored and handled to maintain optimal activity?

For optimal performance and longevity of DNAH7 antibody, HRP conjugated:

• Storage temperature: Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles: Aliquot antibody upon first thaw to minimize degradation

• Working solution: When diluted for use, maintain at 4°C and use within 24 hours

• Buffer conditions: The antibody is typically provided in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Shipping conditions: Typically shipped with ice packs; verify integrity upon arrival

Research has shown that properly stored HRP-conjugated antibodies maintain >90% activity for at least 12 months when stored according to manufacturer recommendations.

How can DNAH7 antibody be applied in primary ciliary dyskinesia (PCD) research?

DNAH7 antibody has proven valuable in PCD research through several methodological approaches:

Diagnostic applications:

• Immunofluorescence microscopy: Enables visualization of DNAH7 localization in respiratory ciliary axonemes

• Correlation with genetic findings: Can confirm functional impact of variants in genes like CCDC39 and CCDC40

Research applications:

• Characterization of IDA components: Studies have used DNAH7 antibodies to demonstrate that defects in the 96 nm axonemal ruler affect assembly of IDA components

• Differential diagnosis: While PCD individuals with CCDC39/CCDC40 variants show absence of DNAH7, those with N-DRC defects (variants in DRC1/CCDC164, CCDC65, GAS8) or ODA defects (DNAH5) show normal DNAH7 distribution

Protocol optimization for PCD samples:

• Collect respiratory epithelial cells via nasal or bronchial brushing

• Fix cells in 4% paraformaldehyde

• Permeabilize with 0.2% Triton X-100

• Block with 5% BSA/normal serum

• Incubate with DNAH7 antibody (1:50-200 dilution)

• Use acetylated α-tubulin as ciliary marker for co-localization studies

• Analyze using high-resolution confocal microscopy

This approach enables researchers to correlate genetic variants with specific alterations in ciliary ultrastructure.

What methodological approaches can resolve contradictory findings when using DNAH7 antibodies?

When faced with contradictory results using DNAH7 antibodies, consider implementing these methodological approaches:

Antibody validation strategies:

• Multiple detection methods: Combine immunofluorescence, Western blotting, and ELISA to verify findings

• Different antibody clones: Test antibodies recognizing different epitopes within DNAH7 (e.g., AA 730-903 versus AA 2301-2400)

• Genetic knockdown validation: Use siRNA to deplete DNAH7 and confirm antibody specificity

Technical optimization:

• Epitope accessibility: Optimize antigen retrieval methods (heat-induced vs. enzymatic)

• Fixation comparison: Test multiple fixation protocols (PFA, methanol, acetone)

• Blocking optimization: Test different blocking agents to reduce background

• Positive controls: Include samples with known DNAH7 expression

• Signal amplification: Consider tyramide signal amplification for low-abundance detection

Analytical approaches:

• Quantitative analysis: Use digital image analysis software for objective quantification

• Co-localization studies: Assess overlap with known interaction partners (e.g., DNALI1)

• Subcellular fractionation: Separate chromatin-bound from soluble fractions before analysis

These approaches have successfully resolved contradictions in studies of DNAH7 localization in PCD patients with different genetic variants .

How can DNAH7 antibodies be leveraged in research on sperm flagella anomalies?

DNAH7 antibodies provide valuable tools for investigating sperm flagella anomalies, particularly in cases of asthenozoospermia and multiple morphological abnormalities of the sperm flagella (MMAF):

Experimental approaches:

• Comparative immunolocalization: Analyze DNAH7 distribution in normal versus abnormal sperm flagella

• Co-localization with other flagellar markers: Use multi-color immunofluorescence to examine spatial relationships with other axonemal components

• Correlative light and electron microscopy: Combine immunolocalization with ultrastructural analysis

Clinical research applications:

• Diagnostic potential: DNAH7 antibody staining patterns can help categorize subtypes of MMAF

• Genotype-phenotype correlations: Compare DNAH7 staining patterns in cases with known variants in DNAH gene family members

• Fertility outcome prediction: Research indicates that infertile males with variants in DNAH7 can achieve successful pregnancy outcomes through ICSI

Protocol considerations:

• Use mild fixation to preserve flagellar structure

• Include membrane permeabilization optimization

• Apply DNAH7 antibody at 1:50-200 dilution for immunofluorescence

• Counterstain with acetylated α-tubulin or other axonemal markers

This approach has helped establish that DNAH7 plays a critical role in sperm flagella beating and male fertility .

What considerations are important when using DNAH7 antibodies in multi-color immunofluorescence experiments?

Multi-color immunofluorescence with DNAH7 antibodies requires careful planning to achieve optimal results:

Experimental design considerations:

• Antibody compatibility: When using DNAH7 antibody with other primary antibodies, ensure they are raised in different host species to avoid cross-reactivity

• Epitope accessibility: Different fixation methods may variably affect epitope accessibility for DNAH7 versus other targets

• Signal intensity balancing: DNAH7 may require signal amplification to match intensity with more abundant proteins

Technical optimization strategies:

• Sequential staining: Consider sequential rather than simultaneous incubation if antibodies are incompatible

• Spectral separation: Ensure fluorophores have minimal spectral overlap (particularly important when using FITC-conjugated DNAH7 antibody)

• Blocking optimization: Use species-specific blocking to minimize cross-reactivity

• Controls: Include single-stained controls for spectral compensation and bleed-through assessment

Validated multi-color combinations:

• DNAH7 antibody works effectively with acetylated α-tubulin for ciliary co-localization studies

• For studying inner dynein arm components, combinations of DNAH7 with DNAH1 and DNAH6 antibodies have been successfully employed

These approaches have enabled researchers to characterize the differential localization of IDA components in respiratory cilia from healthy individuals and PCD patients .

How do DNAH7 mutations impact colorectal cancer research and what methodological approaches can antibodies provide?

Recent research has identified DNAH7 mutations as potentially beneficial for colorectal cancer patients receiving immune checkpoint inhibitors (ICIs) , opening new avenues for research where DNAH7 antibodies can play a crucial role:

Research findings:

• Colorectal cancer patients with DNAH7 mutations showed improved response to ICIs (P<0.05)

• DNAH7 mutation was associated with higher ESTIMATE scores, immune scores, and matrix scores (P<0.001)

• Pathways related to small molecule transport, keratinization, and immune response were enriched in DNAH7 mutated tissues

Methodological approaches using DNAH7 antibodies:

• Tumor microenvironment characterization:

  • Use IHC with DNAH7 antibodies to assess expression patterns in tumor versus normal tissues

  • Compare expression in microsatellite instability-high versus microsatellite stable colorectal cancers

• Predictive biomarker development:

  • Develop immunohistochemical protocols to detect wild-type versus mutated DNAH7 protein

  • Correlate DNAH7 staining patterns with genetic testing results and treatment outcomes

• Mechanistic studies:

  • Use DNAH7 antibodies to identify protein interaction partners in cancer cells

  • Investigate changes in subcellular localization of DNAH7 in cancer versus normal cells

  • Compare DNAH7 protein levels between DNAH7-mutated and wild-type tumors using Western blot

• Protocol recommendations:

  • For paraffin-embedded sections: Use 1:200-400 dilution of DNAH7 antibody

  • For frozen sections: Use 1:100-500 dilution

  • Include appropriate positive controls (respiratory epithelium) and negative controls

These approaches can help elucidate the mechanisms by which DNAH7 mutations influence tumor immunology and response to immunotherapy.

What protein extraction and Western blotting protocols are optimal for detecting DNAH7?

Detecting DNAH7 (~461 kDa) by Western blotting requires specialized protocols optimized for high molecular weight proteins:

Protein extraction protocol:

• Harvest cells or tissue in ice-cold RIPA buffer supplemented with protease inhibitors

• For ciliated cells, consider direct lysis in Laemmli buffer to improve high molecular weight protein recovery

• Sonicate samples briefly (3 × 10s pulses) while keeping on ice

• Centrifuge at 14,000×g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

Western blotting protocol optimized for DNAH7:

• Gel selection: Use NuPAGE 3-8% Tris-acetate gels specifically designed for high molecular weight proteins

• Sample preparation: Denature samples at 70°C for 10 minutes (not higher, to prevent aggregation)

• Electrophoresis: Run at lower voltage (100V) for extended time (3-4 hours)

• Transfer: Use wet transfer to PVDF membrane at low amperage (250mA) overnight at 4°C

• Blocking: Block overnight at 4°C in 5% skim milk in TBS-T

• Primary antibody: Incubate with anti-DNAH7 rabbit polyclonal antibody diluted in 5% skim milk in TBS-T

• Secondary antibody: Incubate with goat anti-rabbit HRP antibody for 1 hour at room temperature

• Detection: Use ECL Prime Western Blotting Detection Reagent with extended exposure times

This protocol has successfully detected DNAH7 as a single specific band at the expected size of approximately 461 kDa in control samples .

How can chromatin immunoprecipitation be optimized using DNAH7 antibodies?

While DNAH7 is primarily known as an axonemal protein, recent studies suggest potential nuclear functions. Optimizing chromatin immunoprecipitation (ChIP) with DNAH7 antibodies requires:

Pre-optimization considerations:

• Antibody validation: First confirm nuclear localization of DNAH7 in your cell type using fractionation and immunofluorescence

• Chromatin binding assessment: Perform chromatin binding assay similar to HRP-3 protocols to verify chromatin association

• Epitope accessibility: Evaluate if the antibody epitope (e.g., AA 730-903 or AA 2301-2400) remains accessible in chromatin-bound DNAH7

ChIP protocol optimization:

• Crosslinking: Test both formaldehyde (1%) and dual crosslinking (DSG followed by formaldehyde)

• Sonication: Optimize sonication conditions for fragments of 200-500bp

• Antibody amount: Typically 2-5μg per ChIP reaction, but titration is recommended

• Chromatin amount: Start with 25μg of chromatin per reaction

• Controls: Include IgG control and positive control antibody (e.g., H3)

• Washing stringency: Optimize salt concentration in wash buffers

qPCR primer design considerations:

• Design multiple primer pairs covering different regions of potential DNAH7 target genes

• Include primers targeting known DNAH7-independent regions as negative controls

• Use similar approach to E2F1 promoter analysis that demonstrated HRP-3 binding