DYNC2H1 Antibody

What is DYNC2H1 and what cellular functions does it have?

DYNC2H1 (Cytoplasmic dynein 2 heavy chain 1) is a large molecular motor protein that plays critical roles in multiple cellular processes. It functions primarily as a motor for intraflagellar retrograde transport, which is essential for cilia biogenesis. The protein is also involved in transport between the endoplasmic reticulum and Golgi apparatus and may participate in Golgi organization within cells . At the molecular level, DYNC2H1 drives retrograde transport of the IFT-A protein complex that regulates tip-to-base transport in cilia, which is crucial for the generation and maintenance of these cellular structures .

What are the key structural domains of DYNC2H1 protein?

The DYNC2H1 protein contains several functionally important domains that contribute to its motor activity:

• Six AAA+ domains forming a hexameric ring-like ATP-hydrolyzing motor domain (AAA1-AAA6)

• AAA1 (amino acids 1651–1875)

• AAA2 (amino acids 1938–2161)

• AAA3 (amino acids 2251–2505)

• AAA4 (amino acids 2617–2863)

• AAA5 (amino acids 3244–3479)

• AAA6 (amino acids 3697–3912)

• A microtubule-binding stalk domain between AAA4 and AAA5 (amino acids 2881–3227)

• An N-terminal tail domain (DHC_N1, amino acids 234–676)

• A linker domain (DHC_N2, amino acids 1120–1520) that changes position in different nucleotide states to create the powerstroke for microtubule motility

• A conserved C-terminal domain arranged on top of the ATPase ring (Dynein_heavy, amino acids 3621–4311)

What applications can DYNC2H1 antibodies be used for?

Available DYNC2H1 antibodies can be used for multiple research applications depending on the specific antibody's validation profile:

These applications allow researchers to study DYNC2H1 expression, localization, and function in various experimental systems .

How can DYNC2H1 antibodies be used to investigate ciliopathies?

DYNC2H1 mutations have been implicated in several ciliopathies, including Jeune asphyxiating thoracic dystrophy (ATD) and short rib polydactyly syndrome (SRP). Researchers can employ DYNC2H1 antibodies to:

• Assess DYNC2H1 protein expression and localization in patient-derived cells

• Evaluate the impact of DYNC2H1 mutations on intraflagellar transport (IFT)

• Visualize DYNC2H1 distribution in ciliated tissues from control and disease models

• Develop imaging of IFT in patient-derived cells as a potential diagnostic tool

Studies have shown that DYNC2H1 is a major cause of JATD (Jeune asphyxiating thoracic dystrophy), particularly affecting North Europeans, and resulting in a predominantly thoracic phenotype. Imaging of intraflagellar transport in patient-derived cells using DYNC2H1 antibodies provides a useful diagnostic approach for this condition .

What are the best methodological approaches for studying DYNC2H1 interaction with other IFT components?

When investigating DYNC2H1 interactions with other intraflagellar transport components, researchers should consider:

• Co-immunoprecipitation (Co-IP): Use DYNC2H1 antibodies to pull down protein complexes from ciliated cell lysates, followed by immunoblotting for suspected interacting partners. This approach requires antibodies suitable for immunoprecipitation applications.

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions in situ when two proteins are within 40 nm of each other. Using DYNC2H1 antibodies in combination with antibodies against other IFT components allows visualization of interaction sites within cells.

• Immunofluorescence co-localization: Double-staining with DYNC2H1 antibodies and antibodies against other IFT proteins can demonstrate spatial co-localization in ciliary structures.

• Live cell imaging: For dynamic studies, consider using cells expressing fluorescently tagged DYNC2H1 in conjunction with immunolabeling of fixed timepoints.

Each approach should include appropriate controls to validate specificity of the observed interactions .

How do mutations in DYNC2H1 affect protein function and localization?

DYNC2H1 mutations have been found throughout the gene, affecting various functional domains. Research using DYNC2H1 antibodies has revealed:

• Missense mutations (e.g., p.Met1991, p.Gln1537, p.Thr1987, p.Gly2461, p.Asp3015, p.Met3762) often affect conserved amino acids across species and are predicted to be damaging to protein function .

• Premature stop codon mutations typically result in truncated proteins with compromised function or protein instability.

• Structural impacts: Some mutations, such as p.R2481Q, can create new hydrogen bonds (e.g., between Q2481 and Y2477) that alter protein conformation and function .

• Localization changes: DYNC2H1 antibody studies in patient-derived cells show that mutations can disrupt normal ciliary localization patterns and affect retrograde IFT.

• Mutation hotspots: Certain regions, particularly within the motor domain, show higher mutation frequency in patient populations, suggesting functional importance.

For functional studies, immunofluorescence with DYNC2H1 antibodies combined with markers of ciliary compartments can help determine how mutations affect proper localization and transport functions .

What are the optimal sample preparation methods for DYNC2H1 antibody applications?

Sample preparation varies by application:

For Western Blot (WB):

• Use fresh tissue or cells when possible, particularly ciliated cells like RPE1 or testis tissue where DYNC2H1 is highly expressed

• Employ lysis buffers containing protease inhibitors to prevent degradation of this large protein

• Use low-percentage SDS-PAGE (5-6%) gels to adequately resolve the 493 kDa protein

• Extend transfer times (overnight at low voltage) to ensure complete transfer of this high molecular weight protein

• Validated dilution range: 1:500-1:3000

For Immunohistochemistry (IHC-P):

• Perform antigen retrieval using TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0)

• Use freshly prepared sections from properly fixed tissues

• Optimize incubation times and temperatures for your specific tissue

• Validated dilution range: 1:50-1:500

For Immunofluorescence (IF/ICC):

• Grow cells on coverslips or chamber slides

• Induce ciliogenesis through serum starvation if studying ciliary localization

• Fix with 4% paraformaldehyde or methanol (optimize for your antibody)

• Permeabilize with 0.1-0.5% Triton X-100

• Block thoroughly to reduce background

• Validated dilution range: 1:200-1:800

How can researchers troubleshoot non-specific binding of DYNC2H1 antibodies?

When experiencing non-specific binding with DYNC2H1 antibodies, consider these troubleshooting approaches:

• Increase blocking time and concentration: Use 5-10% normal serum from the same species as the secondary antibody.

• Titrate antibody concentration: Start with the manufacturer's recommended dilution, then adjust as needed. For DYNC2H1 antibodies, validated ranges are:

  • WB: 1:500-1:3000

  • IHC-P: 1:50-1:500

  • IF/ICC: 1:200-1:800

• Include appropriate controls:

  • Positive control: Tissues known to express DYNC2H1 (e.g., testis, kidney)

  • Negative control: Secondary antibody only

  • Specificity control: Preabsorption with immunizing peptide if available

• Optimize wash conditions: Increase wash duration or detergent concentration in wash buffers.

• For Western blot applications: Confirm specificity using recombinant DYNC2H1 protein as a positive control. Commercial recombinant DYNC2H1 protein fragments have been validated with antibodies at 40-80 ng loading amounts .

• Consider alternative antibodies: If persistent non-specific binding occurs, test antibodies raised against different epitopes of DYNC2H1.

How should researchers interpret DYNC2H1 antibody signals in ciliary studies?

When studying DYNC2H1 in ciliary contexts:

• Expected localization pattern: DYNC2H1 typically shows enrichment at the ciliary base with some distribution along the axoneme, reflecting its role in retrograde IFT. Antibody staining should reveal this characteristic pattern in properly ciliated cells.

• Co-localization markers: Always include established ciliary markers to confirm specificity:

  • Acetylated α-tubulin (axoneme marker)

  • γ-tubulin or pericentrin (basal body marker)

  • IFT88 or IFT140 (IFT particle proteins)

• Temporal considerations: DYNC2H1 localization may change during cilium formation, maintenance, and disassembly. Time-course experiments can provide valuable insights.

• Quantitative assessment: Measure signal intensity along the cilium axis to detect subtle distribution changes between experimental conditions.

• 3D imaging: Use Z-stack acquisition and reconstruction to fully capture DYNC2H1 distribution throughout the three-dimensional ciliary structure.

• Live cell imaging validation: When possible, validate fixed-cell immunofluorescence findings with live imaging of fluorescently tagged DYNC2H1 .

How can DYNC2H1 antibodies be used to study ciliopathy mechanisms?

DYNC2H1 antibodies provide valuable tools for investigating ciliopathy mechanisms:

• Patient-derived cells: Researchers can use DYNC2H1 antibodies to analyze protein expression and localization in cells from patients with suspected ciliopathies. Studies have demonstrated that imaging of intraflagellar transport in patient cells can serve as a diagnostic tool for conditions like Jeune asphyxiating thoracic dystrophy (JATD) .

• Mutation screening support: After identifying potential DYNC2H1 mutations through exome sequencing, researchers can use antibodies to assess the impact on protein expression, stability, and localization. This approach helps determine the pathogenicity of variants of uncertain significance .

• Genotype-phenotype correlations: DYNC2H1 antibody staining patterns in different patient samples can help establish correlations between specific mutation types and cellular phenotypes.

• Tissue-specific expression: Immunohistochemical analysis with DYNC2H1 antibodies in various tissues helps understand disease mechanisms. For example, staining in small intestine tissue has been validated and can provide insights into gastrointestinal manifestations of ciliopathies .

• Therapeutic development: DYNC2H1 antibodies can be used to evaluate the efficacy of potential therapeutic interventions aimed at correcting defective protein function or localization in disease models.

What is the recommended approach for analyzing DYNC2H1 variants in clinical research?

When investigating DYNC2H1 variants in clinical research settings:

• Integrated genomic and protein analysis workflow:

  • Begin with exome/genome sequencing to identify potential DYNC2H1 variants

  • Validate variants with Sanger sequencing

  • Use DYNC2H1 antibodies to assess protein expression, size, and localization

• Variant classification considerations:

  • Location within functional domains (motor domain, microtubule-binding stalk, etc.)

  • Conservation across species

  • In silico prediction tools (e.g., PolyPhen)

  • Protein expression/localization changes detected with antibodies

• Cell-based functional assays:

  • Ciliary morphology assessment using immunofluorescence

  • Intraflagellar transport dynamics

  • DYNC2H1 localization in patient-derived cells

• Technical considerations:

  • Ensure complete coverage of DYNC2H1 exons when using next-generation sequencing

  • Be aware that copy number variations (CNVs) affecting DYNC2H1 may be missed by standard sequencing approaches

  • Follow up with protein-level analysis using validated DYNC2H1 antibodies

Research has shown that mutations in DYNC2H1 are a major cause of JATD, particularly affecting North Europeans and causing a predominantly thoracic phenotype. Effective analysis requires combining genomic and protein-level approaches .

How can DYNC2H1 antibodies be optimized for studying its interaction with the IFT-A complex?

To optimize DYNC2H1 antibody use for studying interactions with the IFT-A complex:

• Epitope selection: Choose antibodies targeting regions of DYNC2H1 that are not involved in IFT-A binding to avoid interference with interaction detection. Antibodies targeting the N-terminal tail domain (amino acids 234-676) or C-terminal domain (amino acids 3621-4311) may be preferable to those targeting the motor domains .

• Proximity-based detection methods:

  • Proximity Ligation Assay (PLA) provides single-molecule detection of DYNC2H1 interaction with IFT-A components

  • Förster Resonance Energy Transfer (FRET) microscopy for detecting nanometer-scale interactions

  • These approaches require antibodies that work under native conditions

• Co-immunoprecipitation optimization:

  • Use mild lysis conditions to preserve native protein complexes

  • Consider crosslinking approaches to stabilize transient interactions

  • Include controls for antibody specificity using DYNC2H1-depleted samples

• Immunofluorescence co-localization analysis:

  • Super-resolution microscopy techniques (STED, STORM, SIM) to resolve spatial relationships beyond the diffraction limit

  • Analyze co-localization quantitatively using Pearson's or Mander's coefficients

  • Include appropriate positive and negative co-localization controls

• Dynamic analysis:

  • Use DYNC2H1 antibodies in combination with live-cell imaging of tagged IFT-A components

  • Establish pulse-chase protocols to track complex movement over time

What are the considerations for using DYNC2H1 antibodies in multi-species comparative studies?

When using DYNC2H1 antibodies across different species:

• Epitope conservation assessment:

  • Align DYNC2H1 sequences from target species to identify conserved regions

  • Select antibodies raised against highly conserved epitopes for cross-species applications

  • Available DYNC2H1 antibodies show validated reactivity with human and mouse samples, but may work with other species based on sequence homology

• Validation strategy:

  • Always validate antibody specificity in each new species

  • Include positive controls (tissues known to express DYNC2H1)

  • Use genetic knockouts or knockdowns as negative controls when available

  • Western blot should show bands at the expected molecular weight (approximately 493 kDa)

• Application-specific considerations:

  • For IHC/IF: Optimize fixation and antigen retrieval for each species/tissue

  • For WB: Adjust lysis conditions for tissue-specific differences

  • For IP: Test antibody binding efficiency in each species

• Quantitative comparisons:

  • Standardize protocols across species to ensure comparable results

  • Use internal loading controls appropriate for each species

  • Account for species-specific differences in DYNC2H1 expression levels

• Technical alternatives:

  • Consider epitope tags for difficult-to-detect orthologs

  • Use multiple antibodies targeting different regions for validation

Why might Western blots with DYNC2H1 antibodies show unexpected band patterns?

When Western blots using DYNC2H1 antibodies produce unexpected results, consider these potential issues and solutions:

Recombinant DYNC2H1 protein fragments can serve as useful positive controls, with validated detection at 40-80 ng loading amounts .

How can researchers distinguish between specific and non-specific signals in DYNC2H1 immunostaining?

To differentiate between specific and non-specific DYNC2H1 antibody signals:

• Essential controls:

  • Secondary antibody-only control to assess background

  • DYNC2H1 knockdown/knockout samples as negative controls

  • Peptide competition/pre-absorption to confirm specificity

  • Multiple antibodies targeting different DYNC2H1 epitopes should show similar patterns

• Pattern assessment:

  • Specific signal should reflect known DYNC2H1 biology (enrichment at ciliary base and along axoneme)

  • Co-localization with established ciliary markers supports specificity

  • Signal should be absent or reduced in non-ciliated cells

  • Staining should be reproducible across multiple samples and experiments

• Technical optimization:

  • Titrate antibody concentrations (IF/ICC: 1:200-1:800; IHC: 1:50-1:500)

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Optimize antigen retrieval for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Increase washing steps to reduce background

• Quantitative approaches:

  • Measure signal-to-noise ratios

  • Compare staining intensity between experimental and control samples

  • Use software tools for unbiased quantification of staining patterns

• Alternative confirmation methods:

  • Validate key findings with genetic approaches (e.g., fluorescently tagged DYNC2H1)

  • Consider super-resolution microscopy for detailed localization studies

How can DYNC2H1 antibodies be integrated with emerging imaging technologies?

DYNC2H1 antibodies can be leveraged with cutting-edge imaging approaches to gain new insights:

• Super-resolution microscopy integration:

  • STORM/PALM: Achieve ~20nm resolution of DYNC2H1 localization within ciliary structures

  • STED microscopy: Visualize DYNC2H1 distribution with enhanced resolution

  • Expansion microscopy: Physically expand specimens to resolve nanoscale DYNC2H1 arrangements

  • These techniques require highly specific antibodies and optimization of sample preparation protocols

• Live-cell dynamics studies:

  • Combine fixed-cell DYNC2H1 antibody staining with live imaging of other IFT components

  • Correlative light and electron microscopy (CLEM) to link DYNC2H1 localization with ultrastructural features

  • High-speed imaging of IFT particles to correlate with DYNC2H1 distribution patterns

• Multiplexed imaging approaches:

  • Cyclic immunofluorescence to visualize DYNC2H1 alongside numerous other proteins

  • Mass cytometry imaging to quantify DYNC2H1 levels in heterogeneous cell populations

  • These approaches allow simultaneous visualization of DYNC2H1 with multiple interaction partners

• Functional imaging integration:

  • Combine DYNC2H1 immunofluorescence with live calcium imaging

  • Correlate DYNC2H1 localization with ciliary signaling events

  • Link antibody staining patterns with functional readouts in disease models

• Computational analysis enhancements:

  • Machine learning algorithms to classify DYNC2H1 distribution patterns

  • Automated tracking of IFT particle movement in relation to DYNC2H1 localization

  • Quantitative image analysis pipelines for high-throughput screening applications

What are promising approaches for studying DYNC2H1 in the context of ciliopathy therapeutics?

DYNC2H1 antibodies can support therapeutic development for ciliopathies through:

• High-throughput screening applications:

  • Develop immunofluorescence-based assays to screen compounds that rescue DYNC2H1 localization defects

  • Quantify changes in DYNC2H1 expression or distribution in response to therapeutic candidates

  • Standardize image analysis pipelines for reproducible quantification

• Patient-derived model systems:

  • Use DYNC2H1 antibodies to characterize patient-derived organoids or iPSC models

  • Track disease-specific changes in DYNC2H1 localization and function

  • Evaluate therapeutic efficacy in restoring normal DYNC2H1 patterns

• Gene therapy assessment:

  • Monitor correction of DYNC2H1 expression following gene therapy approaches

  • Evaluate proper localization of exogenously expressed DYNC2H1

  • Assess functional restoration of intraflagellar transport

• Small molecule and biologics development:

  • Screen for compounds that stabilize mutant DYNC2H1 proteins

  • Identify molecules that enhance residual DYNC2H1 function

  • Develop assays to measure restoration of retrograde IFT

• Biomarker development:

  • Explore DYNC2H1 antibody-based assays as potential biomarkers for disease progression

  • Correlate changes in DYNC2H1 localization with clinical outcomes

  • Develop minimally invasive methods to monitor therapeutic response in patient samples

Research has shown that mutations in DYNC2H1 are a major cause of ciliopathies like JATD, particularly affecting North Europeans. Therapeutic approaches targeting this protein represent a promising avenue for intervention in these disorders .