NDEL1 Antibody

What is NDEL1 and what cellular functions does it perform?

NDEL1 (Nuclear distribution protein nudE-like 1, also known as NUDEL or Mitosin-associated protein 1) is a critical protein required for multiple cellular processes centered around microtubule organization and dynamics. It plays essential roles in:

• Organization of the cellular microtubule array and microtubule anchoring at the centrosome

• Regulation of microtubule organization by targeting the microtubule severing protein KATNA1 to the centrosome

• Positive regulation of dynein activity, potentially enhancing dynein-mediated microtubule sliding by targeting dynein to microtubule plus ends

• Maintenance of Golgi integrity

• Regulation of centripetal motion of secretory vesicles

• Coupling of the nucleus and centrosome

• Neuronal migration during brain development

• Neurite outgrowth regulation (in conjunction with DISC1)

• Facilitation of neurofilament polymerization from individual NEFH and NEFL subunits

• Regulation of lysosome peripheral distribution and ruffled border formation in osteoclasts

Understanding these functions is essential for designing experiments that accurately assess NDEL1 activity and interactions.

What types of NDEL1 antibodies are available for research applications?

Research laboratories can access several types of NDEL1 antibodies, each with specific advantages for different experimental applications:

Polyclonal antibodies provide excellent sensitivity for detecting endogenous levels of protein, while monoclonal antibodies offer superior specificity. Phospho-specific antibodies are invaluable for studying NDEL1 regulation through post-translational modifications .

What is the typical molecular weight of NDEL1 in Western blot applications?

NDEL1 has a calculated molecular weight of approximately 38 kDa, though the apparent molecular weight may vary slightly depending on post-translational modifications and experimental conditions. When performing Western blot analysis, researchers should expect to observe a band between 35-40 kDa representing the full-length protein. Some antibodies may also detect additional bands representing isoforms or post-translationally modified versions of NDEL1 .

To ensure accurate identification of NDEL1:

• Always run appropriate positive controls (e.g., brain tissue lysates)

• Include molecular weight markers

• Validate using genetic approaches (siRNA knockdown or CRISPR knockout) when possible

• Consider the effects of phosphorylation on migration patterns

How should I optimize Western blot conditions for NDEL1 antibodies?

Optimizing Western blot conditions for NDEL1 detection requires attention to several key parameters:

• Sample Preparation:

  • Brain tissue or neuronal cells typically express high levels of NDEL1

  • Use RIPA buffer with protease inhibitors for general extraction

  • Include phosphatase inhibitors when studying phosphorylated forms

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel Electrophoresis:

  • 10-12% SDS-PAGE gels provide optimal resolution for NDEL1 (~38 kDa)

  • Load 10-30 μg of total protein per lane

• Transfer and Blocking:

  • PVDF membranes typically perform well for NDEL1 detection

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody Dilutions:

  • Primary antibody: Start with manufacturer-recommended dilutions (typically 1:500-1:2,000)

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: 1:5,000-1:10,000, incubate for 1 hour at room temperature

• Detection:

  • Both chemiluminescence and fluorescence-based detection systems work well

  • For weak signals, consider enhanced sensitivity substrates or longer exposure times

Always include positive controls (e.g., brain tissue lysates) and negative controls (secondary antibody only) to validate specificity .

What are the recommended protocols for immunohistochemistry using NDEL1 antibodies?

Successful immunohistochemistry with NDEL1 antibodies requires attention to tissue preparation, antigen retrieval, and staining conditions:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde

  • For paraffin embedding, process tissues using standard protocols

  • Section at 4-6 μm thickness for optimal staining

• Antigen Retrieval:

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

  • Boil sections for 10-20 minutes, then cool to room temperature

• Staining Protocol:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol

  • Block non-specific binding with 5-10% normal serum in PBS with 0.1% Triton X-100

  • Incubate with primary antibody at recommended dilutions (typically 1:50-1:200)

  • Optimal incubation: overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibodies

  • Develop with DAB and counterstain with hematoxylin

• Controls:

  • Include positive control tissues (brain sections)

  • Use isotype controls to assess background

  • Consider NDEL1 knockdown tissues as negative controls

NDEL1 typically shows strong expression in neuronal tissues, with specific subcellular localization patterns reflecting its various functions .

How can I validate the specificity of NDEL1 antibodies in my experimental system?

Rigorous validation of NDEL1 antibodies is essential for generating reliable research data. Multiple complementary approaches should be employed:

• Genetic Approaches:

  • siRNA or shRNA knockdown of NDEL1 should reduce signal intensity

  • CRISPR/Cas9 knockout cells provide definitive negative controls

  • The pNDEL1 signal in mouse brain slices was significantly diminished after NDEL1 knockdown by in utero electroporation of an shRNA construct

• Peptide Competition:

  • Pre-incubation of the antibody with excess immunizing peptide should abolish specific staining

  • Non-related peptides should not affect signal

• Multiple Antibody Validation:

  • Use antibodies from different sources targeting different epitopes

  • Compare monoclonal and polyclonal antibodies

  • Compare the staining patterns across different applications

• Signal Specificity Tests:

  • For phospho-specific antibodies, treatment with phosphatases should eliminate signal

  • For phospho-specific antibodies, test on mutants lacking the phosphorylation site

  • The phosphorylation detected by anti-pNDEL1 antibody was decreased in NDEL1 S332A and virtually absent in NDEL1 S336A mutants

• Expected Expression Pattern:

  • NDEL1 should be highly expressed in neural tissues

  • Subcellular localization should be consistent with known biology (centrosome, microtubules, etc.)

Proper validation ensures experimental rigor and reproducibility across different research conditions .

How can I use NDEL1 antibodies to study its phosphorylation patterns and regulatory mechanisms?

Studying NDEL1 phosphorylation provides insights into its regulation during neuronal development and function. Here's a methodological approach:

• Phospho-specific Antibodies:

  • Use antibodies that specifically recognize phosphorylated residues (e.g., anti-pNDEL1 for S336/S332)

  • Confirm specificity with phospho-mutants (S→A) and phospho-mimetics (S→E/D)

  • Validate with phosphatase treatments

• Temporal Expression Analysis:

  • In the developing mouse brain, pNDEL1 signal peaks at embryonic day 18 (E18) and postnatal day 7 (P7), corresponding to stages of residual neuronal migration and intense neurite outgrowth

  • Use immunoblotting of tissues from various developmental stages

  • Quantify relative phosphorylation levels normalized to total NDEL1

• Kinase Identification:

  • NDEL1 is sequentially phosphorylated by the DYRK2-GSK3β complex

  • Use kinase inhibitors to disrupt phosphorylation

  • Co-expression of DYRK2-GSK3β S9A increases endogenous NDEL1 phosphorylation

• Subcellular Localization:

  • Phosphorylated NDEL1 is prominently detected in growth cones

  • Use co-immunostaining with cytoskeletal markers (phalloidin for F-actin, α-tubulin)

  • pNDEL1 overlaps with both F-actin and α-tubulin in filopodia-like structures

• Functional Assays:

  • Compare wild-type NDEL1 with phospho-mutants in neurite outgrowth assays

  • NDEL1 knockdown significantly reduces both total neurite length and longest neurite length by approximately 31% and 33%, respectively

This multi-faceted approach allows for comprehensive characterization of NDEL1 phosphorylation dynamics and functional consequences.

What are the best approaches for studying NDEL1 interactions with cytoskeletal components?

NDEL1 functions at the interface of microtubule and actin cytoskeletal networks. Here are methodological approaches to study these interactions:

• Co-localization Studies:

  • Use high-resolution confocal or super-resolution microscopy

  • Triple-stain for NDEL1, microtubules (tubulin), and actin (phalloidin)

  • Phosphorylated NDEL1 colocalizes with both F-actin and α-tubulin, particularly in filopodia-like structures at growth cones

• Co-immunoprecipitation (Co-IP):

  • IP NDEL1 and probe for cytoskeletal proteins

  • Reverse IP: pull down tubulin or actin and probe for NDEL1

  • Include detergent controls to distinguish direct vs. indirect interactions

  • Use crosslinking approaches for transient interactions

• Proximity Ligation Assay (PLA):

  • Allows detection of protein-protein interactions in situ

  • Useful for detecting NDEL1 interactions with dynein, DISC1, or cytoskeletal components

• Live Cell Imaging:

  • Fluorescently tagged NDEL1 (GFP-NDEL1 or similar constructs)

  • Track co-movement with labeled cytoskeletal components

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics

  • GFP-tagged rat Nde1 or Ndel1 constructs can be generated using PCR amplification of coding sequences

• Structure-Function Analysis:

  • Test deletion mutants of NDEL1 for altered cytoskeletal binding

  • Assess phospho-mutants for their impact on cytoskeletal interactions

  • Use domain-specific antibodies to block particular interaction interfaces

These approaches provide complementary data on the spatial and temporal dynamics of NDEL1-cytoskeleton interactions.

How should I design experiments to study NDEL1's role in neuronal migration and development?

Investigating NDEL1's role in neuronal development requires approaches that span multiple scales, from molecular interactions to cellular behavior:

• In Utero Electroporation:

  • Introduce NDEL1 shRNA, overexpression constructs, or mutants

  • Co-electroporate with fluorescent markers (e.g., GFP)

  • Analyze cortical migration patterns at different developmental stages

  • This technique allows for temporal and spatial control of gene manipulation

• Primary Neuronal Cultures:

  • Isolate neurons from embryonic or postnatal brains

  • Transfect with NDEL1 constructs or knockdown reagents

  • Quantify parameters such as:

    • Neurite length and branching complexity

    • Growth cone morphology and dynamics

    • Neuronal polarization and axon specification

  • NDEL1 knockdown significantly reduces neurite length in cultured hippocampal neurons

• Time-lapse Imaging:

  • Track migrating neurons in slice cultures

  • Monitor cytoskeletal dynamics in growth cones

  • Assess nuclear movement relative to the centrosome

• Biochemical Profiling:

  • Compare phosphorylation patterns across developmental stages

  • Identify stage-specific binding partners through IP-MS

  • The phosphorylation of NDEL1 peaks at E18 and P7, corresponding to critical stages of neuronal development

• Rescue Experiments:

  • After NDEL1 knockdown, attempt to rescue phenotypes with:

    • Wild-type NDEL1

    • Phospho-mutants (S336A, S332A)

    • Phospho-mimetics (S336D, S332D)

    • Domain-specific mutants

  • This approach helps establish the specific functional domains and modifications required for NDEL1's role in development

These experimental designs provide mechanistic insights into how NDEL1 orchestrates neuronal development through its interactions with the cytoskeleton and regulatory proteins.

Why might I observe multiple bands in Western blot when using NDEL1 antibodies?

Multiple bands in NDEL1 Western blots can stem from several biologically relevant or technical factors:

• Biological Factors:

  • Post-translational modifications (particularly phosphorylation)

  • Alternatively spliced isoforms

  • Proteolytic processing in vivo

  • The phosphorylation state can affect migration patterns, particularly for phosphorylated forms recognized by phospho-specific antibodies

• Technical Factors:

  • Sample preparation (proteolytic degradation during extraction)

  • Cross-reactivity with related proteins (e.g., NDE1)

  • Non-specific binding of the antibody

  • Insufficient blocking or washing

To determine the nature of additional bands:

• Isoform verification: Compare with recombinant isoforms or literature reports

• Phosphorylation assessment: Treat samples with phosphatase before SDS-PAGE

• Specificity validation: Use knockdown/knockout samples as negative controls

• Cross-reactivity testing: Pre-absorb antibody with recombinant proteins

• Optimization: Adjust antibody concentration, blocking conditions, and washing steps

When interpreting multiple bands, consider the developmental stage and tissue type, as NDEL1 expression and modification patterns change during development .

What controls are essential when using phospho-specific NDEL1 antibodies?

Phospho-specific antibodies require rigorous controls to ensure reliable and interpretable results:

• Validation Controls:

  • Phospho-mutant constructs (S→A): Should show reduced or absent signal

  • The phosphorylation detected by anti-pNDEL1 antibody was decreased in NDEL1 S332A and virtually absent in NDEL1 S336A mutants

  • Phospho-mimetic constructs (S→D/E): May show altered recognition

  • Lambda phosphatase treatment: Should eliminate signal from phosphorylated samples

• Biological Controls:

  • Developmental series: NDEL1 phosphorylation peaks at specific developmental stages (E18, P7)

  • Kinase manipulation: Overexpression of DYRK2-GSK3β increases phosphorylation

  • Kinase inhibitor treatments: Should reduce phosphorylation in a dose-dependent manner

• Technical Controls:

  • Total NDEL1 antibody on parallel blots/sections for normalization

  • Loading controls (GAPDH, β-actin) to ensure equal protein loading

  • Secondary-only controls to assess background

  • Blocking peptide competition to confirm specificity

• Experimental Design Controls:

  • Include both positive tissues (brain) and negative tissues (low-expressing)

  • Include multiple time points when studying dynamic processes

  • Run parallel assays with general and phospho-specific antibodies

Proper controls ensure that observed signals genuinely represent the phosphorylated form of NDEL1 rather than technical artifacts or cross-reactivity .

How can I interpret changes in NDEL1 localization during neuronal development?

NDEL1 exhibits dynamic localization patterns during neuronal development that reflect its changing functions:

• Developmental Timeline Analysis:

  • Embryonic stages: NDEL1 is critical for neuronal migration

  • Postnatal stages: NDEL1 functions in neurite outgrowth and synaptogenesis

  • Adult brain: NDEL1 maintains cytoskeletal integrity

  • The phosphorylation of NDEL1 peaks at embryonic day 18 (E18) and postnatal day 7 (P7), corresponding to stages of neuronal migration and neurite outgrowth

• Subcellular Localization Patterns:

  • Centrosomal localization: Associated with neuronal migration and cell division

  • Growth cone enrichment: Indicates role in neurite extension

    • Phosphorylated NDEL1 is prominent in growth cones and filopodia-like structures

    • Co-localizes with both F-actin and microtubules at growth cones

  • Dendritic localization: May indicate roles in dendritic arborization

  • Synaptic localization: Suggests functions in synaptic plasticity

• Correlation with Functional States:

  • NDEL1 knockdown reduces neurite length, suggesting its localization to growth cones is functionally significant

  • Changes in phosphorylation state correlate with changes in localization pattern

  • Interaction with different partners (DISC1, LIS1, dynein) may trigger relocalization

• Experimental Approach:

  • Use co-localization studies with markers for specific subcellular structures:

    • Pericentrin for centrosomes

    • Phalloidin for F-actin in growth cones

    • MAP2 for dendrites

    • Synaptic markers (PSD-95, synaptophysin)

  • Quantify co-localization coefficients across developmental stages

  • Correlate localization changes with functional assays

These interpretive frameworks help researchers connect NDEL1 localization patterns to its various functions throughout neuronal development .

How can NDEL1 antibodies be used to study neurodevelopmental disorders?

NDEL1 antibodies offer powerful tools for investigating neurodevelopmental disorders, particularly those involving neuronal migration defects or cytoskeletal abnormalities:

• Disorder-Associated Mutations:

  • Generate antibodies against common NDEL1 mutants or modified forms

  • Compare localization and expression patterns between normal and pathological states

  • NDEL1 interacts with DISC1, a protein implicated in schizophrenia and other psychiatric disorders

• Patient-Derived Samples:

  • Analyze NDEL1 expression and phosphorylation in postmortem brain tissues

  • Examine NDEL1 in patient-derived neurons (from iPSCs)

  • Look for altered NDEL1-cytoskeleton interactions

• Animal Models:

  • Use NDEL1 antibodies to characterize phenotypes in genetic models

  • Assess developmental abnormalities in cortical layering

  • Evaluate neurite outgrowth and neuronal morphology defects

  • Monitor phosphorylation patterns throughout development

• Therapeutic Development:

  • Screen compounds that normalize NDEL1 phosphorylation or localization

  • Use phospho-specific antibodies to monitor treatment efficacy

  • Develop assays for high-throughput screening

• Biomarker Potential:

  • Evaluate whether NDEL1 or its modified forms could serve as biomarkers

  • Develop more sensitive antibodies for detecting low abundance forms

These approaches leverage antibody technologies to bridge fundamental NDEL1 biology with clinical neurodevelopmental disorders research .

What are the most promising techniques for examining NDEL1 dynamics in live cells?

Advanced imaging techniques combined with specific labeling strategies offer unprecedented insights into NDEL1 dynamics:

• Fluorescent Fusion Proteins:

  • GFP-NDEL1 constructs allow visualization of NDEL1 trafficking

  • Rat Nde1 or Ndel1 coding sequences can be PCR-amplified and inserted into pEGFP-C1 vectors

  • Photoactivatable or photoconvertible tags for pulse-chase experiments

  • Split fluorescent proteins to visualize protein-protein interactions

• FRET/FLIM Applications:

  • Design FRET pairs to monitor NDEL1 interactions with binding partners

  • Develop FRET-based sensors for NDEL1 phosphorylation state

  • Use FLIM to distinguish bound vs. unbound states

• Super-Resolution Microscopy:

  • STORM/PALM imaging for nanoscale localization

  • SIM for improved resolution of cytoskeletal interactions

  • Lattice light-sheet microscopy for 3D dynamics with reduced phototoxicity

• Optogenetic Approaches:

  • Light-inducible dimerization to control NDEL1 interactions

  • Optogenetic control of kinases that phosphorylate NDEL1

  • Spatiotemporal control of NDEL1 activity in specific subcellular compartments

• Quantitative Analysis Methods:

  • Single-particle tracking of NDEL1 molecules

  • Correlation analysis with cytoskeletal dynamics

  • Machine learning approaches for pattern recognition in complex datasets

These emerging techniques will help answer key questions about how NDEL1 dynamically interacts with the cytoskeleton and regulatory proteins during neuronal development and function .