DYNC2LI1 Antibody

What is DYNC2LI1 and why is it important in research?

DYNC2LI1, also known as D2LIC, LIC3, or CGI-60, is a 351-352 amino acid cytoplasmic protein belonging to the dynein light intermediate chain family . It functions as a motor for intraflagellar retrograde transport and plays a crucial role in cilia biogenesis . The protein exists in five alternatively spliced isoforms and is a key component of the cytoplasmic dynein-2 complex, which likely consists of a DYNC2H1 homodimer and several DYNC2LI1 light intermediate chains .

DYNC2LI1 is important in research because it's required for correct intraflagellar transport (IFT), the bidirectional movement of particles necessary for the assembly, maintenance, and functioning of primary cilia . Additionally, it's involved in regulating ciliary length . Mutations in this gene can result in several types of ciliopathies, including short-rib thoracic dysplasia 15 with polydactyly (SRTD15) , making it a significant target for studying human developmental disorders.

What applications are DYNC2LI1 antibodies suitable for?

DYNC2LI1 antibodies have been validated for multiple research applications:

• Western Blotting (WB): Most commercially available DYNC2LI1 antibodies are validated for WB at dilutions ranging from 1:500 to 1:2000

• Immunohistochemistry (IHC): Both paraffin-embedded and frozen section protocols

• Immunocytochemistry/Immunofluorescence (ICC/IF): Typically used at concentrations of 1-4 μg/ml

• Immunoprecipitation (IP): Especially with monoclonal antibodies like H-4

• Enzyme-linked immunosorbent assay (ELISA)

The choice of application should be guided by the specific research question and the validation data provided by the antibody manufacturer for each application.

How should DYNC2LI1 antibodies be stored for optimal performance?

For optimal antibody performance and longevity, follow these storage recommendations:

• Short-term storage (up to 1 month): Store at 4°C

• Long-term storage: Aliquot and store at -20°C

• Avoid freeze-thaw cycles as they can degrade antibody quality and performance

• Most DYNC2LI1 antibodies are provided in PBS with preservatives such as sodium azide and stabilizers like glycerol (typically 40-50%)

When handling antibodies for experiments, it's best to thaw only the amount needed and keep the stock on ice while working to minimize degradation due to temperature fluctuations.

What species reactivity do commercial DYNC2LI1 antibodies exhibit?

Based on the available search results, commercial DYNC2LI1 antibodies show reactivity to:

• Human DYNC2LI1

• Mouse DYNC2LI1

• Rat DYNC2LI1

When selecting an antibody, it's important to check the specific species reactivity data provided by the manufacturer. Some antibodies are specifically designed with high sequence identity to orthologs, such as the one mentioned in result , which has 82% sequence identity to mouse and 86% to rat orthologs.

What is the difference between polyclonal and monoclonal DYNC2LI1 antibodies?

Polyclonal antibodies like PA5-64075 and NBP2-38008 are often generated against recombinant proteins containing significant portions of DYNC2LI1 , while monoclonal antibodies like H-4 target specific epitopes and may offer more consistent results across different experiments .

How can researchers validate the specificity of DYNC2LI1 antibodies?

Validating antibody specificity is critical for reliable experimental results. For DYNC2LI1 antibodies, consider these approaches:

• Knockdown/knockout verification: Use siRNA or CRISPR-Cas9 to reduce DYNC2LI1 expression and confirm corresponding reduction in antibody signal

• Recombinant protein controls: Compare detection of recombinant DYNC2LI1 versus related proteins to assess cross-reactivity

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide prior to use in experiments; specific signals should be blocked

• Multiple antibody verification: Use antibodies targeting different epitopes of DYNC2LI1 and compare results

• Protein array testing: Some manufacturers validate antibodies on protein arrays containing the target protein plus hundreds of non-specific proteins to ensure specificity

• Multiple application concordance: Verify that results are consistent across different detection methods (e.g., WB, IF, IHC)

In one documented approach, specificity testing involved analysis of extracts from various cell lines using DYNC2LI1 antibody at 1:1000 dilution with secondary HRP Goat Anti-Rabbit IgG detection .

What are the optimal experimental conditions for studying DYNC2LI1 in cilia using immunofluorescence?

When studying DYNC2LI1 in cilia using immunofluorescence techniques, consider these optimized conditions:

• Fixation: Standard 4% paraformaldehyde fixation preserves both ciliary structure and DYNC2LI1 localization

• Permeabilization: Mild detergents (0.1-0.2% Triton X-100) maintain ciliary structures while allowing antibody access

• Antibody concentration: For immunofluorescence, use DYNC2LI1 antibodies at 1-4 μg/ml concentration

• Co-staining markers: Include ciliary markers such as acetylated α-tubulin or ARL13B to identify cilia structures

• Controls: Include cells with DYNC2LI1 knockdown as negative controls

• Confocal imaging: Use confocal microscopy to precisely localize DYNC2LI1 within ciliary structures

• Cilia induction: Serum starvation (24-48 hours) typically maximizes cilia formation in cell cultures before staining

Research has shown that in control conditions, cilia have a mean length of approximately 1.83 μm, while DYNC2LI1-depleted cells show shorter cilia (approximately 1.42 μm) with abnormally broadened ciliary tips in a significant percentage of cells (14% vs. 6% in controls) .

How do DYNC2LI1 mutations affect cilia, and how can antibodies help investigate these effects?

DYNC2LI1 mutations result in abnormal cilia formation and function:

• Ciliary morphology changes: Mutations lead to shorter cilia (approximately 1.42 μm compared to 1.83 μm in controls) with abnormally bulged tips, similar to other ciliopathies with retrograde IFT defects

• Retrograde transport defects: Impaired intraflagellar transport leads to accumulation of proteins at ciliary tips

• Disease manifestations: Mutations can cause short-rib thoracic dysplasia 15 with polydactyly (SRTD15)

DYNC2LI1 antibodies can help investigate these effects through:

• Protein localization studies: Immunofluorescence microscopy to determine abnormal DYNC2LI1 distribution in mutant cells

• Protein-protein interaction analysis: Immunoprecipitation to examine how mutations affect DYNC2LI1 interactions with other dynein-2 components

• Expression level assessment: Western blotting to determine if mutations affect DYNC2LI1 protein stability or expression

• Tissue distribution analysis: IHC to compare DYNC2LI1 distribution in normal versus affected tissues

• Developmental studies: Tracking DYNC2LI1 expression during development, as it shows differential expression between fetal and adult tissues, with significantly higher levels in fetal brain, lung, and kidney

What approaches can be used to study DYNC2LI1 interactions with DYNC2H1?

Recent structural and biochemical studies have revealed that DYNC2LI1 interacts with DYNC2H1 through specific domains:

• Co-immunoprecipitation (Co-IP): Using antibodies against either DYNC2LI1 or DYNC2H1 to pull down the complex and analyze interactions

  • In published experiments, lysates from HEK293T cells co-expressing DYNC2H1(N)-mCherry and DYNC2LI1-EGFP constructs were immunoprecipitated with GST-fused anti-mCherry nanobodies

• Domain mapping: Research has shown that both the N-terminal Ras-like G domain and the C-terminal coil region (residues 318-352) of DYNC2LI1 participate in its interaction with DYNC2H1

• Truncation constructs: Creating DYNC2LI1 constructs with truncations from the C-terminus helps analyze which regions are essential for DYNC2H1 interaction

• Yeast two-hybrid assays: Can be used to screen for specific interaction domains

• Proximity ligation assay (PLA): For visualizing DYNC2LI1-DYNC2H1 interactions in situ within cells

• Structural studies: The cryo-EM structure of the dynein-2 complex provides insights into how DYNC2LI1 and DYNC2H1 interact

Understanding these interactions is critical as they form the basis of dynein-2 complex assembly and function in retrograde intraflagellar transport.

How can researchers distinguish between the five DYNC2LI1 isoforms in their experiments?

DYNC2LI1 exists as five alternatively spliced isoforms , which presents challenges for researchers:

It's worth noting that mutations discovered in patients with ciliopathies are located in the two longest isoforms, suggesting these have particular pathogenic significance in skeletal development .

What controls should be included when using DYNC2LI1 antibodies for ciliary studies?

When studying ciliary function using DYNC2LI1 antibodies, include these critical controls:

• Negative controls:

  • Primary antibody omission

  • Isotype controls (matching IgG from the same species)

  • DYNC2LI1 knockdown or knockout cells

  • Pre-immune serum for polyclonal antibodies

• Positive controls:

  • Cells/tissues known to express DYNC2LI1 (e.g., brain, kidney, lung, testes which show high expression levels)

  • Recombinant DYNC2LI1 protein

• Specificity controls:

  • Blocking peptide competition (pre-incubating antibody with immunizing peptide)

  • Multiple antibodies targeting different epitopes

• Technical controls:

  • Ciliary markers (acetylated α-tubulin, ARL13B) to identify cilia

  • Basal body markers (γ-tubulin, pericentrin) to identify ciliary base

  • Nuclear counterstain (DAPI) for cell identification

• Experimental controls:

  • Time course of ciliogenesis (e.g., after serum starvation)

  • Comparison of cilia parameters (length, morphology) between wild-type and experimental conditions

• Validation across techniques:

  • Correlate immunofluorescence findings with biochemical data (western blot, IP)

  • Confirm protein expression with mRNA expression

Published research shows that comparing scrambled controls to DYNC2LI1-depleted cells can reveal significant differences in cilia length (1.83 μm vs. 1.42 μm) and morphology (6% vs. 14% abnormal ciliary structures) .

What are the optimal dilutions and protocols for Western blotting with DYNC2LI1 antibodies?

For optimal Western blotting results with DYNC2LI1 antibodies, follow these guidelines:

• Antibody dilutions:

  • Primary antibody: 1:500 to 1:2000 (commonly 1:1000)

  • Secondary antibody: 1:10,000 for HRP-conjugated anti-rabbit IgG

• Sample preparation:

  • Protein loading: 25 μg protein per lane is typically sufficient

  • Lysis buffer: Standard RIPA buffer with protease inhibitors

• Blocking conditions:

  • 3-5% nonfat dry milk in TBST has been successfully used

  • Alternative: 5% BSA in TBST for phospho-specific applications

• Detection systems:

  • ECL (Enhanced Chemiluminescence) Basic Kit works well for visualization

  • Typical exposure time: 5 seconds for standard applications

• Controls:

  • Positive control: Lysates from cells known to express DYNC2LI1

  • Negative control: Lysates from DYNC2LI1 knockdown cells

  • Loading control: β-actin, GAPDH, or similar housekeeping proteins

• Special considerations:

  • DYNC2LI1 has five isoforms, so multiple bands may be observed

  • The two longest isoforms are predominantly expressed in most tissues

How can DYNC2LI1 antibodies be used to study ciliopathies in patient samples?

DYNC2LI1 antibodies can provide valuable insights when studying ciliopathies in patient samples:

• Patient tissue immunohistochemistry:

  • Compare DYNC2LI1 localization in patient vs. control tissues

  • Assess ciliary morphology using co-staining with ciliary markers

  • Examine tissue-specific expression patterns, particularly in affected tissues

• Patient-derived cells:

  • Primary fibroblasts or induced pluripotent stem cells (iPSCs) from patients

  • Quantify changes in cilia length, which is typically reduced in DYNC2LI1 mutations (1.42 μm vs. 1.83 μm in controls)

  • Assess percentage of cells with bulged ciliary tips (14% in DYNC2LI1-depleted cells vs. 6% in controls)

• Molecular diagnosis:

  • Correlate DYNC2LI1 protein expression with genetic testing results

  • Evaluate effect of specific mutations on protein localization and stability

• Functional studies:

  • Complement genetic studies with protein-level analyses

  • Assess retrograde IFT by tracking intraflagellar cargo accumulation

  • Examine DYNC2LI1 interactions with other dynein-2 components

• Therapeutic development:

  • Monitor restoration of normal ciliary function in response to experimental therapies

  • Use as biomarkers for treatment response

• Developmental timing considerations:

  • DYNC2LI1 shows differential expression between fetal and adult tissues

  • Higher expression levels occur in fetal brain, lung, and kidney tissues

What is the significance of the DYNC2LI1 N-terminal G domain in experimental design?

The N-terminal Ras-like G domain of DYNC2LI1 has special significance for experimental design:

• Structural role: This domain is evolutionarily conserved among light intermediate chains of both dynein-1 and dynein-2 complexes

• Protein-protein interactions: The G domain mediates critical interactions with DYNC2H1, as demonstrated by:

  • X-ray crystallographic structure of thermophilic yeast dynein-1 LIC

  • Biochemical experiments with human DYNC1LI1

  • Cryo-EM structure of the dynein-2 complex

• Experimental considerations:

  • Antibodies targeting this region may interfere with DYNC2LI1-DYNC2H1 interactions

  • When designing truncation constructs, preserve the integrity of this domain

  • For interaction studies, include both the G domain and C-terminal coil region (residues 318-352)

• Mutation analysis: Many pathogenic mutations may affect this domain, altering binding to DYNC2H1

• Immunoprecipitation strategy: For co-IP experiments studying dynein-2 complex formation, consider:

  • Using antibodies that don't interfere with G domain interactions

  • Testing C-terminal tagged constructs that preserve N-terminal interactions

Understanding the structural and functional significance of this domain is essential for properly designing experiments to study DYNC2LI1 function and interactions.

What are the current limitations in DYNC2LI1 antibody research?

Despite significant progress, several limitations exist in current DYNC2LI1 antibody research:

• Isoform specificity: Most commercial antibodies don't distinguish between the five alternatively spliced isoforms

• Species cross-reactivity: While antibodies recognize human, mouse, and rat DYNC2LI1, there's limited validation for other model organisms

• Tissue-specific differences: Expression levels vary across tissues and developmental stages , but antibody performance in different tissue contexts is underreported

• Post-translational modifications: Limited information on how antibodies perform against modified forms of DYNC2LI1

• Structural constraints: Antibodies that might disrupt protein-protein interactions can complicate functional studies

• Quantitative applications: Few antibodies are validated for quantitative applications like ELISA

Future antibody development should address these limitations, particularly focusing on isoform-specific antibodies and validation across diverse experimental conditions.

What emerging techniques might enhance DYNC2LI1 research beyond traditional antibody applications?

Emerging technologies promise to advance DYNC2LI1 research beyond conventional antibody applications:

• CRISPR knock-in tagging: Endogenous tagging of DYNC2LI1 to avoid antibody specificity issues and overexpression artifacts

• Super-resolution microscopy: Techniques like STORM, PALM, or STED to visualize DYNC2LI1 localization within ciliary subcompartments at nanometer resolution

• Live-cell imaging: Using split-GFP or HaloTag approaches to track DYNC2LI1 dynamics in living cells

• Proximity labeling: BioID or APEX2 fusions to map the DYNC2LI1 interactome in different cellular contexts

• Single-molecule techniques: To study the biophysical properties of DYNC2LI1 in dynein-2 motor function

• Cryo-electron tomography: For structural studies of DYNC2LI1 within intact cilia

• Organoid models: To study DYNC2LI1 function in more physiologically relevant 3D tissue contexts

• Patient-derived iPSCs: To model ciliopathies and test therapeutic approaches targeting DYNC2LI1 function