DNAH7 Antibody, Biotin conjugated

What is DNAH7 and why is it a significant target for antibody-based detection in research?

DNAH7 (Dynein, Axonemal, Heavy Chain 7) is an axonemal dynein protein crucial for ciliary and flagellar motility in eukaryotic cells. This protein belongs to the dynein heavy chain family, which forms the inner and outer dynein arms that power microtubule sliding in cilia and flagella. DNAH7 is particularly important in studying ciliopathies, male infertility conditions, and respiratory disorders involving ciliary dysfunction .

The detection of DNAH7 using specific antibodies allows researchers to:

• Localize the protein in tissues with ciliated epithelium (e.g., fallopian tubes, respiratory tract)

• Study protein expression patterns in normal vs. pathological conditions

• Investigate protein-protein interactions within the axonemal complex

• Analyze the consequences of DNAH7 mutations or downregulation

What are the key differences between biotin-conjugated and unconjugated DNAH7 antibodies?

Biotin-conjugated DNAH7 antibodies offer several methodological advantages compared to their unconjugated counterparts:

The biotin-conjugated DNAH7 antibody (e.g., ABIN7150878) contains covalently attached biotin molecules that bind with high affinity to streptavidin or avidin detection systems, eliminating the need for species-specific secondary antibodies .

What experimental applications are biotin-conjugated DNAH7 antibodies validated for?

Validated Applications:

• ELISA: Optimal working dilution should be determined by the investigator

Potential Applications (requiring optimization):

• Immunohistochemistry: May be suitable using streptavidin-HRP or streptavidin-fluorophore detection

• Flow cytometry: For detection of intracellular DNAH7

• Immunoprecipitation: Using streptavidin beads for pull-down assays

• ChIP assays: When studying transcription factors associated with DNAH7 expression

Note that while unconjugated DNAH7 antibodies have been validated for multiple applications including ICC, IF, IHC, and Western blot , the biotin-conjugated version specifically requires validation for these applications in your experimental system.

How should biotin-conjugated DNAH7 antibodies be properly stored and handled to maintain optimal activity?

Proper storage and handling of biotin-conjugated DNAH7 antibodies is critical for maintaining their functionality:

Storage Conditions:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as this can damage the antibody and reduce performance

• Aliquot the stock solution into smaller volumes before freezing to minimize freeze-thaw cycles

Handling Recommendations:

• When working with the antibody, keep it on ice or at 4°C

• Return to storage promptly after use

• The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 preservative

• Note the safety precaution: The product contains Proclin, which is classified as a poisonous and hazardous substance requiring handling by trained staff only

Working Solution Preparation:

• Dilute only the amount needed for immediate use

• Use sterile, protein-free buffers for dilution

• Prepare fresh working solutions for each experiment

What optimization strategies should be employed when using biotin-conjugated DNAH7 antibodies in streptavidin-based detection systems?

When utilizing biotin-conjugated DNAH7 antibodies in streptavidin-based detection systems, researchers should implement the following optimization strategies:

Blocking Endogenous Biotin:

• Pre-block endogenous biotin in samples using an avidin/biotin blocking kit

• Use tissues fixed in aldehyde-based fixatives rather than Bouin's solution (which preserves endogenous biotin)

• Implement a 15-30 minute blocking step with unconjugated avidin followed by d-biotin

Titration Optimization:

• Perform a detailed titration series (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

• Assess signal-to-noise ratio at each dilution

• Select the dilution providing maximum specific signal with minimal background

Detection System Selection:

The optimal working dilution should be determined empirically for each experimental system and application .

How can researchers validate the specificity of biotin-conjugated DNAH7 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable results. For biotin-conjugated DNAH7 antibodies, implement these validation approaches:

Essential Validation Methods:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (human DNAH7 protein, AA 730-903)

  • Run parallel assays with blocked and unblocked antibody

  • Specific binding should be significantly reduced in the peptide-blocked sample

• Genetic Models:

  • Use DNAH7 knockout/knockdown models as negative controls

  • Test antibody reactivity in DNAH7-overexpression systems

  • Example: Test in Zmynd10-/- mice where DNAH7 is downregulated

• Multi-antibody Validation:

  • Compare results with alternative DNAH7 antibodies targeting different epitopes (e.g., AA 2301-2400)

  • Consistent localization/expression patterns support specificity

• Appropriate Controls:

  • No primary antibody control

  • Isotype control (rabbit IgG-biotin)

  • Tissue-specific positive controls (e.g., fallopian tube samples known to express DNAH7)

  • Tissue-specific negative controls (tissues without ciliated cells)

• Western Blot:

  • Confirm the antibody detects a protein of the expected molecular weight (~461 kDa for full-length DNAH7)

  • Multiple bands may indicate degradation products or alternative splice variants

Successfully validated antibodies should show consistent patterns across multiple detection methods and model systems.

What are the optimal antigen retrieval methods when using biotin-conjugated DNAH7 antibodies for immunohistochemistry?

While the biotin-conjugated DNAH7 antibody has not been explicitly validated for immunohistochemistry, related DNAH7 antibodies provide guidance on optimal antigen retrieval methods:

Heat-Induced Epitope Retrieval (HIER) Methods:

Enzymatic Retrieval Methods (Alternative Approach):

• Proteinase K (10-20 μg/mL, 10-15 minutes at room temperature)

• May preserve tissue morphology better but potentially lower sensitivity

For paraffin-embedded sections, HIER with pH 6.0 citrate buffer is the recommended starting point for DNAH7 detection . After retrieval, implement a streptavidin-biotin blocking step to minimize endogenous biotin interference prior to antibody application.

How do different fixation methods affect epitope recognition by biotin-conjugated DNAH7 antibodies?

Fixation methods significantly impact epitope integrity and accessibility for DNAH7 antibodies. The following table summarizes the effects of common fixatives:

For cultured cells expressing DNAH7:

• Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

• Perform mild permeabilization (0.1-0.3% Triton X-100, 5-10 minutes)

• Implement thorough blocking of non-specific binding sites

For FFPE tissue sections, limit fixation time to 24-48 hours and implement appropriate antigen retrieval methods to maximize epitope accessibility while maintaining tissue morphology.

What troubleshooting approaches can address common challenges when using biotin-conjugated DNAH7 antibodies?

When working with biotin-conjugated DNAH7 antibodies, researchers may encounter several technical challenges. The following troubleshooting guide addresses common issues:

High Background Signal:

Weak or Absent Signal:

Inconsistent Results:

• Standardize all protocol steps (fixation, antigen retrieval, incubation times)

• Prepare consistent antibody dilutions from master stocks

• Include appropriate positive and negative controls in each experiment

• Document all experimental conditions thoroughly

When troubleshooting, change only one variable at a time and maintain appropriate controls to identify the source of the issue.

What are the recommended protocols for using biotin-conjugated DNAH7 antibodies in chromogenic immunohistochemistry?

While the biotin-conjugated DNAH7 antibody is primarily validated for ELISA , the following protocol provides a foundation for adapting it to chromogenic immunohistochemistry:

Sample Preparation and Deparaffinization:

• Section FFPE tissues at 4-5 μm thickness

• Deparaffinize with xylene (3 × 5 minutes)

• Rehydrate through graded alcohols (100%, 95%, 70%, 50%) to water

Antigen Retrieval:

• Perform HIER using citrate buffer (pH 6.0) for 20 minutes

• Allow sections to cool in buffer for 20 minutes

• Wash in PBS or TBS (3 × 5 minutes)

Endogenous Blocking:

• Block endogenous peroxidase (3% H₂O₂, 10 minutes)

• Block endogenous biotin using avidin/biotin blocking kit

• Perform protein blocking (2-5% BSA or normal serum, 30-60 minutes)

Antibody Incubation:

• Apply biotin-conjugated DNAH7 antibody at optimized dilution (start with 1:50-1:200)

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly in PBS or TBS (3 × 5 minutes)

Detection:

• Apply streptavidin-HRP conjugate (1:100-1:500 dilution)

• Incubate for 30-60 minutes at room temperature

• Wash thoroughly (3 × 5 minutes)

• Develop with DAB or other appropriate substrate

• Counterstain with hematoxylin, dehydrate, and mount

Controls to Include:

• Positive control (human fallopian tube tissue showing ciliated epithelium)

• Negative control (omission of primary antibody)

• Peptide competition control

The working dilution should be empirically determined through titration experiments for optimal signal-to-noise ratio .

How can researchers optimize dual immunofluorescence protocols involving biotin-conjugated DNAH7 antibodies?

Dual immunofluorescence using biotin-conjugated DNAH7 antibodies requires careful planning and optimization:

Protocol Optimization Strategies:

• Sequential vs. Simultaneous Approach:

  • Sequential: Apply and detect biotin-DNAH7 antibody first, followed by the second primary antibody

  • Simultaneous: Apply both primary antibodies together, then detect sequentially

  • Recommendation: Sequential approach minimizes cross-reactivity issues

• Fluorophore Selection:

  • Choose spectrally distinct fluorophores (minimal overlap)

  • Consider tissue autofluorescence characteristics

  • Recommended combination: Streptavidin-Alexa Fluor 488 for DNAH7 with red-emitting fluorophore for second target

• Validation Controls:

  • Single-label controls for each antibody

  • Secondary-only controls

  • Absorption controls with immunizing peptides

  • Cross-reactivity controls to ensure detection systems don't interact

Successful dual immunofluorescence will allow co-localization analysis of DNAH7 with other proteins of interest in ciliated tissues or cells.

What are the critical considerations when using biotin-conjugated DNAH7 antibodies for detecting low-abundance targets?

Detecting low-abundance DNAH7 expression requires enhanced sensitivity approaches:

Signal Amplification Methods:

Protocol Modifications:

• Extended Incubation Times:

  • Increase primary antibody incubation to 48-72 hours at 4°C

  • Use humidified chambers to prevent drying

• Optimized Buffer Compositions:

  • Add 0.1% Triton X-100 to enhance tissue penetration

  • Include 5% polyethylene glycol as an enhancing agent

• Sample Preparation Considerations:

  • Use thinner sections (3-4 μm) for improved antibody penetration

  • Consider non-formalin fixatives that better preserve epitopes

• Microscopy Optimization:

  • Use high-NA objectives and sensitive detection systems

  • Implement image acquisition with extended exposure times

  • Apply deconvolution algorithms to improve signal-to-noise ratio

These approaches should be implemented systematically, with appropriate controls at each step to distinguish specific signal from amplified background.

How do biotin-conjugated and unconjugated DNAH7 antibodies compare in Western blot applications?

While the biotin-conjugated DNAH7 antibody has not been specifically validated for Western blot, comparative analysis with unconjugated DNAH7 antibodies provides insights:

Comparative Analysis for Western Blot Applications:

Protocol Adaptations for Biotin-Conjugated Antibody:

• Include avidin in blocking buffer to sequester endogenous biotin

• Use streptavidin-HRP at 1:1000-1:5000 dilution

• Consider extended exposure times for detection of low-abundance DNAH7

• Include positive control lysates from tissues known to express DNAH7 (testis)

Western blot detection of DNAH7 has been successfully demonstrated using unconjugated antibodies in mouse testis extracts, showing downregulation in Zmynd10-/- mice .

What experimental designs can determine the functional significance of DNAH7 in ciliary motility using the biotin-conjugated antibody?

Investigating DNAH7's functional role in ciliary motility requires integrated experimental approaches:

Immunolocalization Studies:

• Use biotin-conjugated DNAH7 antibody to localize the protein within axonemal structures

• Co-localize with markers of outer dynein arms (e.g., DNAH5) and inner dynein arms (e.g., DNAH1)

• Compare localization in normal vs. pathological samples (e.g., primary ciliary dyskinesia)

Interaction Studies:

• Proximity Ligation Assay (PLA):

  • Combine biotin-DNAH7 antibody with antibodies against potential interacting proteins

  • Use streptavidin and appropriate secondary antibodies conjugated to PLA probes

  • Fluorescent signals indicate protein-protein proximity (<40 nm)

• Co-Immunoprecipitation:

  • Use streptavidin beads to pull down biotin-DNAH7 antibody complexes

  • Analyze precipitated proteins by mass spectrometry or Western blot

  • Validate interactions through reciprocal co-IP experiments

Functional Correlation:

• Correlate DNAH7 expression/localization with:

  • Ciliary beat frequency measurements

  • High-speed video microscopy of ciliary waveform

  • Electron microscopy of axonemal ultrastructure

• Experimental model systems:

  • Human respiratory epithelial cells (air-liquid interface cultures)

  • Sperm flagella

  • Multiciliated ependymal cells

Knockdown/Knockout Approaches:

• Generate DNAH7 knockdown models using siRNA or CRISPR-Cas9

• Confirm knockdown efficiency using the biotin-conjugated DNAH7 antibody

• Assess phenotypic consequences on ciliary structure and function

These integrated approaches can establish the specific role of DNAH7 in axonemal dynein arm assembly and ciliary motility.

How should researchers design experiments to validate DNAH7 antibody specificity across different species?

Validating DNAH7 antibody specificity across species requires systematic cross-reactivity testing:

Cross-Reactivity Analysis Framework:

• Sequence Alignment Analysis:

  • Align the immunogen sequence (human DNAH7 AA 730-903) with DNAH7 orthologs from target species

  • Calculate percent identity and similarity

  • Identify conserved epitopes within the immunogenic region

  Species	Percent Identity with Human DNAH7 (AA 730-903)	Predicted Cross-Reactivity
  Mouse	Not specified in search results	Validated
  Rat	91% identity predicted	Predicted
  Dog, Cow, Sheep, Pig, Horse, Chicken, Rabbit	Not specified	Predicted for some DNAH7 antibodies

• Stepwise Validation Protocol:

  • Begin with Western blot analysis of lysates from multiple species

  • Confirm band at expected molecular weight (~461 kDa)

  • Test immunohistochemistry on ciliated tissues from different species

  • Compare with species-specific positive controls (e.g., known DNAH7 antibodies)

• Knockout/Knockdown Validation:

  • Test antibody in DNAH7 knockout/knockdown models from different species

  • Confirm signal reduction/elimination in genetic models

• Absorption Controls:

  • Pre-absorb antibody with recombinant DNAH7 proteins from different species

  • Test remaining reactivity to determine cross-reactivity specificity

• Developmental/Tissue Expression Profiling:

  • Compare antibody staining patterns with known DNAH7 expression profiles

  • Consistent with expected ciliated tissue distribution across species

For the biotin-conjugated DNAH7 antibody, although predominantly validated for human samples , evidence suggests it may cross-react with mouse DNAH7 based on reactivity of related antibodies .

How should researchers address potential artifacts and misinterpretations when using biotin-conjugated DNAH7 antibodies?

Researchers must be vigilant about potential artifacts and implement strategies to ensure accurate interpretation:

Common Artifacts and Mitigation Strategies:

Verification Approaches:

• Multi-method Confirmation:

  • Verify DNAH7 expression using alternative methods (RT-qPCR, RNA-seq)

  • Compare protein localization using antibodies targeting different epitopes

  • Correlate with known expression patterns from databases (Human Protein Atlas)

• Biological Correlation:

  • Confirm DNAH7 localization in ciliated structures using electron microscopy

  • Verify expected co-localization with other axonemal components

  • Demonstrate functional correlations (e.g., altered expression in ciliopathies)

• Documentation and Reporting:

  • Document all experimental conditions thoroughly

  • Include representative images of positive and negative controls

  • Report criteria used to distinguish specific from non-specific staining

  • Disclose image processing methods and parameters