dnai7 Antibody

What are the validated applications for DNAI7 antibodies?

DNAI7 antibodies have been rigorously validated for several applications in molecular and cellular biology research. Based on current literature and manufacturer specifications, DNAI7 antibodies are suitable for Western Blot (WB), Immunohistochemistry with paraffin-embedded tissues (IHC-P), and Immunocytochemistry (ICC) .

For Western blotting applications, optimal results are typically achieved using a dilution of 1/500, as demonstrated with rat brain tissue lysate samples. The predicted band sizes for DNAI7 detection are 25 kDa, 53 kDa, and 83 kDa, with an observed band size of approximately 84 kDa in experimental settings .

For IHC-P applications, a 1/100 dilution has shown effective staining in human lymph node tissue samples .

What is the species reactivity profile of DNAI7 antibodies?

Most commercially available DNAI7 antibodies demonstrate reactivity with human and rat samples . The protein sequence homology between species is significant, with mouse and rat DNAI7 sharing approximately 84% identity with human DNAI7 . This high degree of conservation makes many anti-human DNAI7 antibodies suitable for cross-species applications, though optimization may be required for specific experimental settings.

How do you optimize protein extraction for DNAI7 detection?

For optimal DNAI7 detection, consider the following methodological approach:

• Use freshly prepared tissue or cell lysates whenever possible

• Include protease inhibitors in your lysis buffer to prevent protein degradation

• For membrane-associated proteins like DNAI7, employ a lysis buffer containing mild detergents (0.5-1% NP-40 or Triton X-100)

• Perform tissue homogenization or cell lysis at 4°C to minimize proteolysis

• Centrifuge lysates at high speed (>10,000 × g) to remove cellular debris

• Quantify protein concentration using Bradford or BCA assay prior to loading for Western blot analysis

For Western blotting, reducing conditions are recommended based on experimental evidence with DNAI7 detection .

How can nonspecific binding be reduced in DNAI7 antibody applications?

Nonspecific binding is a common challenge with DNA-conjugated antibodies, including those targeting DNAI7. Research has shown that nonspecific binding is particularly problematic in nuclear regions and can vary depending on the antibody . To minimize this issue, consider implementing the following optimization strategies:

• Convert single-stranded conjugated DNA to double-stranded DNA by hybridizing complementary DNA strands before antibody incubation

• Add dextran sulfate (0.02-0.5%) to compete for electrostatic interactions with positively charged molecules

• Include sheared salmon sperm DNA (100-500 μg/ml) as a blocking agent

• Increase buffer ionic strength by adding NaCl (100-500 mM)

• Use high molecular weight (>500 kDa or 9-20 kDa) dextran sulfate for more consistent performance

A comparative study demonstrated that these modifications significantly reduced nonspecific nuclear signals while preserving specific target signals in immunofluorescence applications .

What are the recommended fixation methods for DNAI7 detection in tissue samples?

For optimal DNAI7 detection in tissue samples, fixation method selection is critical. The following approach is recommended based on current research practices:

• For IHC-P applications: 10% neutral buffered formalin fixation for 24-48 hours, followed by paraffin embedding

• For frozen sections: 4% paraformaldehyde fixation for 10-15 minutes

• For cultured cells: 4% paraformaldehyde (10 minutes) or methanol/acetone (1:1, -20°C, 10 minutes)

Post-fixation antigen retrieval methods such as heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) may enhance staining intensity and specificity for formalin-fixed samples .

How can DNAI7 antibodies be validated for research applications?

Rigorous validation of DNAI7 antibodies is essential for ensuring experimental reliability. A comprehensive validation approach includes:

• Specificity testing:

  • Knockdown/knockout controls using siRNA or CRISPR-Cas9

  • Peptide competition assays with the immunogen

  • Western blot analysis confirming expected molecular weight(s)

• Application-specific validation:

  • For WB: Confirm band size (observed ~84 kDa) matches predictions (25, 53, and 83 kDa)

  • For IHC/ICC: Compare staining patterns with known subcellular localization

  • Perform parallel testing with multiple antibody clones when available

• Cross-species reactivity assessment:

  • Test antibody performance in samples from different species

  • Consider sequence homology (84% identity between human and mouse/rat)

• Documentation:

  • Maintain detailed records of all validation experiments

  • Document lot-to-lot variability when replacing antibody stocks

What is the biological function of DNAI7 in cilia regulation?

DNAI7 (CASC1) is involved in cilia function regulation through its association with the multisubunit axonemal dynein complex . Axonemal dyneins are force-generating proteins that produce movement in respiratory cilia, similar to other dynein family members that generate force toward the minus ends of microtubules .

Key functional aspects of DNAI7 include:

• Participation in the regulation of cilia motility and structure

• Potential involvement in cell cycle regulation

• Association with microtubule-dependent cellular processes

• Possible roles in intracellular transport mechanisms

Understanding these functions is crucial for research into ciliopathies and related disorders affecting ciliary function.

How can DNAI7 antibodies be incorporated into multiplexed imaging protocols?

Recent advances in multiplexed protein imaging offer powerful approaches for studying DNAI7 in complex cellular contexts. DNA-barcoded antibodies combined with hybridization chain reaction (HCR) methods provide a framework for multiplexed detection .

To implement DNAI7 antibodies in multiplexed protocols:

• Antibody conjugation:

  • Conjugate DNAI7 antibodies with DNA barcodes using validated chemical protocols

  • For optimal results, use shorter DNA sequences (9-10 nucleotides) which show less nonspecific binding than longer sequences

• Protocol optimization:

  • Follow optimized staining protocols that include complementary DNA strands and dextran sulfate

  • Adjust dextran sulfate concentration (optimally 0.02-0.05%) to minimize nonspecific binding

• Signal amplification:

  • Implement HCR for signal amplification when detecting low-abundance targets

  • Select appropriate HCR hairpin pairs based on experimental design (up to 13 pairs available in recent protocols)

• Imaging considerations:

  • Employ sequential imaging or spectrally distinct fluorophores

  • Consider photobleaching strategies for iterative detection of multiple targets

This approach enables visualization of DNAI7 alongside other proteins of interest in both cultured cells and tissue sections .

What are the major challenges in detecting DNAI7 in primary cilia?

Detection of DNAI7 in primary cilia presents several technical challenges:

• Low abundance:

  • DNAI7 may be expressed at relatively low levels in some cell types

  • Signal amplification strategies may be necessary for reliable detection

• Structural complexity:

  • Cilia are complex structures with densely packed proteins

  • Antibody accessibility to target epitopes may be limited

• Fixation sensitivity:

  • Ciliary structures can be disrupted by harsh fixation methods

  • Mild fixation protocols (e.g., 2% PFA for 10 minutes) may better preserve ciliary architecture

• Background issues:

  • Nonspecific antibody binding can obscure true signals, particularly with DNA-conjugated antibodies

  • Implementation of optimized blocking protocols is essential

• Validation concerns:

  • Proper controls should include cells lacking primary cilia (e.g., through IFT88 knockdown)

  • Co-staining with established ciliary markers (acetylated tubulin, ARL13B) helps confirm ciliary localization

How should experiments be designed to study DNAI7 interactions with dynein complex components?

To investigate DNAI7 interactions with other dynein complex components, consider implementing the following experimental design:

• Co-immunoprecipitation (Co-IP):

  • Use anti-DNAI7 antibodies to pull down protein complexes

  • Analyze precipitated proteins by Western blot or mass spectrometry

  • Include appropriate controls (IgG, lysate input)

• Proximity ligation assay (PLA):

  • Combine DNAI7 antibodies with antibodies against potential interacting partners

  • Visualize protein-protein interactions in situ with single-molecule sensitivity

  • Quantify interaction signals using appropriate imaging software

• FRET/BRET analysis:

  • Generate fluorescent/bioluminescent fusion proteins with DNAI7 and potential partners

  • Measure energy transfer as an indicator of protein proximity

  • Validate results with mutant constructs lacking interaction domains

• Cross-linking mass spectrometry:

  • Apply protein cross-linking reagents to stabilize transient interactions

  • Digest complexes and analyze by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Yeast two-hybrid or mammalian two-hybrid screening:

  • Screen for novel DNAI7 interaction partners

  • Validate candidates with orthogonal methods (Co-IP, PLA)

What controls are essential when using DNAI7 antibodies in research applications?

Robust experimental design requires appropriate controls to ensure valid interpretation of results:

• Negative controls:

  • Primary antibody omission

  • Isotype-matched control antibodies

  • DNAI7 knockdown or knockout samples (siRNA, shRNA, CRISPR)

  • Secondary antibody-only controls

• Positive controls:

  • Tissues/cells known to express DNAI7 (e.g., ciliated respiratory epithelium)

  • Recombinant DNAI7 protein for Western blot standardization

  • DNAI7 overexpression systems

• Experimental validation controls:

  • Multiple antibodies targeting different DNAI7 epitopes

  • Peptide competition assays

  • Cross-species validation in tissues with known conservation

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Nuclear counterstains for microscopy (DAPI, Hoechst)

  • Ciliary markers for co-localization (acetylated tubulin, ARL13B)

Inclusion and proper documentation of these controls significantly enhances the reliability and reproducibility of research findings.

How can quantitative analysis of DNAI7 expression be optimized in research settings?

For accurate quantitative analysis of DNAI7 expression, consider these methodological approaches:

• Western blot quantification:

  • Use validated loading controls appropriate for your experimental system

  • Implement linear range detection methods (avoid saturation)

  • Apply normalization to total protein using stain-free technology or Ponceau S

  • Employ densitometry software with background subtraction

• qRT-PCR analysis:

  • Design primers specific to DNAI7 (consider isoform specificity)

  • Validate primer efficiency using standard curves

  • Select appropriate reference genes based on experimental conditions

  • Apply the ΔΔCt or standard curve method for quantification

• Immunofluorescence quantification:

  • Use consistent image acquisition parameters

  • Apply automated analysis workflows to minimize bias

  • Measure integrated density or mean fluorescence intensity

  • Normalize to cell number or area as appropriate

• Flow cytometry:

  • Optimize antibody concentration using titration experiments

  • Include fluorescence-minus-one (FMO) controls

  • Measure median fluorescence intensity for population analysis

  • Consider cell cycle phase when interpreting results

Each of these approaches has strengths and limitations that should be considered based on the specific research questions being addressed.

How can researchers address weak or absent signals in DNAI7 detection?

When encountering weak or absent signals in DNAI7 detection, consider the following troubleshooting approaches:

• Antibody-related factors:

  • Verify antibody quality and storage conditions

  • Test increased antibody concentration

  • Try alternative antibody clones or lots

• Sample preparation:

  • Optimize protein extraction method for your specific tissue/cell type

  • Use freshly prepared samples when possible

  • Include protease inhibitors during extraction

• Detection enhancement:

  • Implement signal amplification methods (HRP polymers, tyramide signal amplification)

  • For IHC/ICC, optimize antigen retrieval methods (HIER, enzymatic retrieval)

  • Extend primary antibody incubation time (overnight at 4°C)

• Protocol modifications:

  • Adjust blocking conditions to reduce background

  • Optimize buffer composition (detergent concentration, salt concentration)

  • Modify washing steps (duration, number of washes)

• Technical considerations:

  • Verify detection system functionality with positive control antibodies

  • Check for instrument sensitivity issues in imaging systems

  • Consider target abundance and expression level variability

What strategies can address nonspecific binding and high background in DNAI7 immunostaining?

Nonspecific binding is a significant challenge in DNAI7 immunostaining, particularly with DNA-conjugated antibodies. The following approaches have demonstrated efficacy in reducing background:

• Blocking optimization:

  • Use 5-10% serum from the species of secondary antibody origin

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 for membrane permeabilization

  • Include 1-3% BSA to reduce nonspecific protein interactions

• DNA-conjugated antibody specific strategies:

  • Pre-block with complementary DNA oligonucleotides at 10x molar excess

  • Add 0.02-0.05% dextran sulfate (high molecular weight >500 kDa preferred)

  • Include 100-500 μg/ml sheared salmon sperm DNA

• Washing modifications:

  • Increase washing duration and frequency

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Use higher salt concentration in wash buffers (150-500 mM NaCl)

• Antibody dilution optimization:

  • Perform antibody titration experiments to determine optimal concentration

  • Prepare antibody dilutions in blocking buffer rather than basic buffer

• Tissue/cell preparation:

  • Optimize fixation conditions (duration, fixative concentration)

  • Implement appropriate antigen retrieval methods

  • Consider tissue-specific autofluorescence quenching methods

A systematic approach testing these variables can significantly improve signal-to-noise ratio in DNAI7 detection .

How can researchers distinguish between specific and nonspecific bands in DNAI7 Western blots?

Distinguishing specific from nonspecific bands in DNAI7 Western blots requires systematic validation:

• Size verification:

  • Compare observed band size (approximately 84 kDa) with predicted sizes (25, 53, and 83 kDa)

  • Consider post-translational modifications that may alter migration

  • Use protein size ladder with appropriate range coverage

• Control experiments:

  • Run parallel lanes with DNAI7 knockdown/knockout samples

  • Include recombinant DNAI7 protein as positive control

  • Perform peptide competition assays with immunizing peptide

• Sample preparation optimization:

  • Test multiple lysis buffers with different detergent compositions

  • Compare reducing vs. non-reducing conditions

  • Include phosphatase inhibitors to preserve post-translational modifications

• Protocol improvements:

  • Optimize transfer conditions for high molecular weight proteins

  • Adjust blocking time and concentration

  • Test different membrane types (PVDF vs. nitrocellulose)

• Technical considerations:

  • Use freshly prepared samples and reagents

  • Ensure consistent loading with appropriate controls

  • Consider gradient gels for better resolution of target protein

Methodical application of these approaches facilitates accurate identification of specific DNAI7 bands .