DNAH7 Antibody, FITC conjugated

What is DNAH7 and why is it significant in cancer research?

DNAH7 (Dynein Axonemal Heavy Chain 7) is a component of the axonemal dynein complex involved in cellular motility. Recent research has established DNAH7 as a significant factor in colorectal cancer (CRC) pathophysiology and treatment response. DNAH7 mutations have been identified as potential biomarkers for response to immune checkpoint inhibitors (ICIs) in CRC patients. Studies involving 690 clinical cohort patients and TCGA data demonstrated that CRC patients with DNAH7 mutations showed significantly improved outcomes when treated with ICIs (P<0.05) . The significance extends beyond simple mutation status, as DNAH7 appears to modulate the tumor immune microenvironment, with mutated samples showing higher ESTIMATE scores, immune scores, and matrix scores compared to wild-type samples (P<0.001) .

What applications are most appropriate for FITC-conjugated DNAH7 antibodies?

FITC-conjugated DNAH7 antibodies are optimized for several fluorescence-based applications:

• Immunofluorescence in cultured cells (IF-ICC) - Recommended dilution 1:50-200

• Immunofluorescence with paraffin-embedded sections (IF-P) - Recommended dilution 1:50-200

• Immunofluorescence with frozen sections (IF-F) - Recommended dilution 1:50-200

• Flow cytometry for detection of DNAH7 expression in cell populations

The direct fluorescent conjugation eliminates the need for secondary antibody incubation, reducing protocol time and potential cross-reactivity issues in multi-labeling experiments.

How should researchers validate DNAH7 antibody specificity for their experimental system?

Methodological validation of DNAH7 antibodies requires a multi-step approach:

• Positive and negative tissue controls: Use tissues with known DNAH7 expression levels. Colorectal tissues are appropriate positive controls based on DNAH7's documented role in CRC .

• Western blot validation: Prior to immunofluorescence applications, confirm specific binding at the expected molecular weight (DNAH7 is approximately 300 kDa).

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (synthetic peptide derived from human DNAH7) to confirm signal abolishment.

• Knockout/knockdown controls: Where possible, use DNAH7 knockout or knockdown cells to confirm antibody specificity.

• Cross-reactivity assessment: Test reactivity against related dynein family proteins, particularly in experimental systems expressing multiple dynein heavy chains.

What is the optimal protocol for using FITC-conjugated DNAH7 antibodies in immunofluorescence studies?

Paraffin-embedded tissue sections protocol:

• Deparaffinization and rehydration:

  • Xylene: 2 × 10 minutes

  • 100% ethanol: 2 × 5 minutes

  • 95%, 80%, 70% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Maintain at 95-98°C for 15-20 minutes

  • Cool to room temperature (20 minutes)

• Permeabilization:

  • 0.2% Triton X-100 in PBS for 10 minutes at room temperature

• Blocking:

  • 5% normal serum in PBS with 0.1% Tween-20 for 1 hour

• Primary antibody incubation:

  • Apply FITC-conjugated DNAH7 antibody (1:50-200 dilution)

  • Incubate overnight at 4°C in a humidified chamber

  • Shield from light to prevent photobleaching

• Nuclear counterstaining:

  • DAPI (1 μg/mL) for 5 minutes

• Mounting:

  • Anti-fade mounting medium

For multi-labeled experiments, carefully select fluorophores with minimal spectral overlap with FITC to reduce bleed-through.

How can researchers effectively use DNAH7 antibodies to investigate colorectal cancer immune microenvironment?

To investigate the relationship between DNAH7 mutation status and the tumor immune microenvironment, a comprehensive approach should include:

This approach has successfully demonstrated that DNAH7 mutations correlate with enrichment of immune-related pathways including allograft rejection, autoimmune thyroid disease, and asthma (P<0.05) .

What controls are essential when using DNAH7 antibodies for research studies?

How do DNAH7 mutations affect response to immune checkpoint inhibitors in colorectal cancer?

DNAH7 mutations significantly enhance the clinical benefit of immune checkpoint inhibitors in colorectal cancer patients. This effect appears to operate through multiple mechanisms:

• Immune microenvironment modulation: Patients with DNAH7 mutations exhibit higher ESTIMATE scores, immune scores, and matrix scores compared to wild-type patients (P<0.001), indicating a more immunologically active tumor environment .

• Pathway enrichment: Gene Set Enrichment Analysis shows that DNAH7 mutations are associated with enrichment of immune-related pathways, including:

  • Allograft rejection

  • Autoimmune thyroid disease

  • Asthma

  • Small molecule transport

• Association with key genes: DNAH7 mutations correlate with expression changes in immune-modulating genes including AQP8, MS4A12, GUCA2B, and ZG16 (P<0.01) .

• Drug susceptibility patterns: DNAH7 mutations demonstrate distinct patterns of drug response, particularly affecting the Druggable Genome. Strong associations were observed with the RTK-RAS pathway and NOTCH pathway in CRC samples .

These findings suggest DNAH7 mutation status could serve as a biomarker for patient selection in immunotherapy clinical trials for colorectal cancer.

What methodological approaches can detect DNAH7 mutations in clinical samples?

Detection of DNAH7 mutations requires a comprehensive molecular approach:

• Next-generation sequencing (NGS):

  • Targeted panel including DNAH7 and related pathway genes

  • Whole exome sequencing for broader mutation landscape

  • Analysis focus on missense mutations, which appear particularly relevant to ICI response

• Digital droplet PCR:

  • For detection of specific recurring mutations

  • Higher sensitivity for low mutation abundance

• Immunohistochemical approach:

  • Use validated DNAH7 antibodies to screen for expression changes

  • Correlation with mutation status through parallel sequencing

  • Recommended dilutions: 1:200-400 for IHC-P; 1:100-500 for IHC-F

• Validation methodology:

  • Sanger sequencing confirmation of detected mutations

  • RNA sequencing to confirm expression changes

  • Protein-level validation with antibody-based methods

Researchers should note that the clinical cohort studies establishing DNAH7 as a biomarker utilized a combination of these approaches to ensure reliable mutation detection .

How can researchers investigate the relationship between DNAH7 and the RTK-RAS pathway?

The documented association between DNAH7 mutations and the RTK-RAS pathway can be investigated through:

• Co-immunoprecipitation studies:

  • Use DNAH7 antibodies to pull down potential protein complexes

  • Western blot analysis for key RTK-RAS pathway components

  • Recommended antibody concentration: 1 μg/μL

• Pathway analysis in mutant vs. wild-type models:

  • Compare phosphorylation states of pathway components

  • Assess downstream transcriptional targets

  • Evaluate pathway activation following perturbation

• Protein-protein interaction (PPI) network analysis:

  • Utilize the STRING online tool with a credibility score threshold >0.4

  • Visualize network models with Cytoscape (V3.7.2)

  • Apply the Maximal Clique Centrality (MCC) algorithm to determine key nodes

• Functional validation:

  • CRISPR-mediated DNAH7 mutation introduction

  • Assessment of RTK-RAS pathway activity changes

  • Drug susceptibility alterations in engineered models

How should researchers quantify DNAH7 expression in immunofluorescence studies?

Quantification of DNAH7 expression from FITC-conjugated antibody studies requires rigorous methodological approaches:

• Image acquisition standardization:

  • Maintain consistent exposure settings across all samples

  • Capture multiple fields (minimum 5-10) per sample

  • Include calibration controls in each imaging session

• Signal quantification methods:

  • Mean fluorescence intensity (MFI) measurement

  • Cell-by-cell analysis for heterogeneity assessment

  • Subcellular localization pattern analysis

• Normalization strategies:

  • Background subtraction using isotype control values

  • Internal reference protein co-staining

  • Cell number normalization for tissue sections

• Data representation:

  • Box-and-whisker plots for population distribution

  • Correlation plots with clinical parameters

  • Integration with mutation data where available

• Statistical analysis:

  • Non-parametric tests for comparing expression levels

  • Correlation coefficients for relationship with other markers

  • Survival analysis based on expression quartiles

What bioinformatic approaches are appropriate for analyzing DNAH7 mutation data?

Analysis of DNAH7 mutation data in cancer research contexts should employ:

• Mutation classification:

  • Categorize by mutation type (missense, nonsense, frameshift)

  • Predict functional impact using tools like SIFT and PolyPhen

  • Map mutations to protein domains and functional regions

• Pathway enrichment analysis:

  • Gene Set Enrichment Analysis (GSEA) methodology

  • Utilize Molecular Signatures Database (MSigDB) gene sets

  • Focus on DNA damage repair, immune response, and cancer-related pathways

• Tumor microenvironment analysis:

  • ESTIMATE algorithm application for stromal and immune scores

  • Correlation with mutation status and clinical outcomes

  • Integration with expression data from the same samples

• Visualization techniques:

  • Mutation lollipop plots mapped to protein domains

  • Heatmaps of associated gene expression changes

  • Clinical outcome Kaplan-Meier plots stratified by mutation status

How can researchers address poor signal-to-noise ratio with FITC-conjugated DNAH7 antibodies?

When encountering weak signal or high background with FITC-conjugated DNAH7 antibodies:

• Optimize antibody concentration:

  • Test dilution series around recommended ranges (1:50-200)

  • Include positive controls at each dilution

  • Determine optimal signal-to-noise ratio empirically

• Enhance antigen retrieval:

  • Extended retrieval times (up to 30 minutes)

  • Alternative buffers (Tris-EDTA pH 9.0 vs. citrate pH 6.0)

  • Enzymatic retrieval options for challenging tissues

• Reduce autofluorescence:

  • Treatment with sodium borohydride (0.1% for 5 minutes)

  • Sudan Black B (0.1% in 70% ethanol for 20 minutes)

  • Commercial autofluorescence quenching reagents

• Improve signal detection:

  • Optimize microscope settings (gain, exposure)

  • Use spectral unmixing for overlapping signals

  • Consider signal amplification methods if direct conjugation is insufficient

• Storage and handling optimization:

  • Maintain antibody at recommended storage conditions (with 50% glycerol buffer)

  • Aliquot stock solution to avoid freeze-thaw cycles

  • Protect from light during all procedures

What are the critical quality control parameters for DNAH7 antibody experiments?

Quality control for DNAH7 antibody experiments should include:

• Antibody validation metrics:

  • Lot-to-lot consistency verification

  • Titration curves to confirm linear response range

  • Cross-reactivity assessment with related proteins

• Technical performance indicators:

  • Signal-to-noise ratio quantification

  • Coefficient of variation across technical replicates

  • Stability assessment under experimental conditions

• Biological validation parameters:

  • Comparison of staining patterns with published data

  • Correlation with mRNA expression where available

  • Consistency of subcellular localization patterns

• Documentation requirements:

  • Complete antibody information (clone, lot, concentration)

  • Detailed experimental protocols

  • Representative images of controls and experimental samples

• Reproducibility assessment:

  • Inter-observer scoring consistency

  • Repeatability across independent experiments

  • Cross-platform validation where possible