DAPB1 Antibody

What is DAB1 protein and why is it important in neuroscience research?

DAB1 is an adaptor protein essential for neuronal migration and maturation in response to the extracellular protein Reelin. It functions by docking to the intracellular part of Reelin receptors (very low density lipoprotein receptor and apoE receptor type 2) and undergoes tyrosine phosphorylation following binding of Reelin to cortical neurons. The DAB1 protein contains a 180-amino acid N-terminal protein interaction/phosphotyrosine-binding (PTB) domain that interacts with NPXY motifs in receptor cytoplasmic tails. DAB1 is crucial for proper brain development, as it mediates the effects of Reelin in establishing the correct positioning and layering of neurons. The phosphorylation of DAB1 is a critical step in signaling pathways that guide neuronal positioning during development .

What types of DAB1 antibodies are available for research applications?

Researchers can utilize several types of DAB1 antibodies depending on their experimental needs:

• Polyclonal antibodies: Such as rabbit polyclonal DAB1 antibody (e.g., 31459-1-AP), which targets multiple epitopes on the DAB1 protein and shows reactivity with human and pig samples .

• Monoclonal antibodies: Including mouse monoclonal antibodies like DAB1 Antibody (G-5), which detects DAB1 protein from mouse, rat, and human origins with high specificity .

• Conjugated antibodies: DAB1 antibodies are available in various conjugated forms including:

  • Agarose conjugates for immunoprecipitation

  • Horseradish peroxidase (HRP) conjugates for direct detection

  • Fluorescent conjugates including phycoerythrin (PE), fluorescein isothiocyanate (FITC), and Alexa Fluor® variants for immunofluorescence applications

The choice between polyclonal and monoclonal antibodies depends on the specific research goals, with polyclonals offering broader epitope recognition and monoclonals providing higher specificity.

What molecular weights should be expected when detecting DAB1 in Western blots?

When performing Western blot analysis of DAB1, researchers should be aware of potential variations in the observed molecular weights:

The variability in observed molecular weights (ranging from 36 to 120 kDa in mouse embryonic samples) likely reflects different isoforms, post-translational modifications, or proteolytic processing of DAB1 . When troubleshooting Western blots, researchers should consider these multiple bands as potentially representing authentic DAB1 protein rather than non-specific binding.

What are the optimal dilutions and conditions for using DAB1 antibodies in various applications?

The optimal working conditions for DAB1 antibodies vary by application and manufacturer. Here is a methodological guide based on validated protocols:

It is crucial to optimize these conditions for each specific experimental setup. For storage, most DAB1 antibodies remain stable for one year when stored at -20°C in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Smaller aliquots (e.g., 20μl) may contain 0.1% BSA for additional stability.

How can researchers validate the specificity of DAB1 antibodies in their experimental systems?

Validating antibody specificity is critical for reliable results. For DAB1 antibodies, a comprehensive validation approach includes:

• Positive and negative controls:

  • Use known DAB1-expressing tissues (pig cerebellum, MCF-7 cells) as positive controls

  • Include DAB1-knockout or siRNA-treated samples as negative controls

• Multiple detection methods:

  • Compare results from Western blotting, immunofluorescence, and immunoprecipitation

  • Observe expected subcellular localization (primarily cytoplasmic for DAB1)

• Cross-reactivity assessment:

  • Test on multiple species if working across evolutionary boundaries

  • Verify reactivity with recombinant DAB1 proteins

• Phosphorylation-specific validation:

  • For phospho-DAB1 antibodies, treat samples with phosphatases as negative controls

  • Use Reelin stimulation to increase DAB1 phosphorylation as a positive control

• Molecular weight verification:

  • Confirm expected molecular weights (45-80 kDa range for native DAB1)

  • Be aware that DAB1 can present as multiple bands (36-120 kDa)

What are the most effective protocols for detecting DAB1 in neuronal tissues with immunohistochemistry?

For optimal DAB1 detection in neuronal tissues:

• Tissue preparation:

  • For fixed tissues: 4% paraformaldehyde fixation for 24h is recommended

  • For frozen sections: Flash-freeze tissues and prepare 10-20μm sections

• Antigen retrieval:

  • Heat-mediated antigen retrieval using citrate buffer (pH 6.0)

  • For DAB1 phospho-epitopes, add phosphatase inhibitors to all buffers

• Blocking and permeabilization:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Permeabilize with 0.1-0.3% Triton X-100 for cytoplasmic epitopes

• Antibody incubation:

  • Primary antibody: Use 1:200-1:800 dilution of DAB1 antibody

  • Incubate overnight at 4°C

  • Secondary antibody: Fluorescent or HRP-conjugated, 1-2h at room temperature

• Multi-label co-localization studies:

  • Combine DAB1 with markers for neurons (MAP2), axons (NFL), and other components of the Reelin pathway

  • This is particularly useful when studying the ApoER2-DAB1 signaling pathway

• Visualization and analysis:

  • For brightfield: DAB (3,3'-diaminobenzidine) substrate for HRP-conjugated antibodies

  • For fluorescence: Use appropriate filters for fluorophore detection

  • Confocal microscopy is recommended for co-localization studies

When examining neurodegenerative conditions, researchers should look for abnormal DAB1 accumulation in swollen, dystrophic neurites surrounding ApoE-enriched neuritic plaques, which is often observed in Alzheimer's disease cases .

How does disruption in DAB1 signaling contribute to neurodegenerative disorders?

DAB1 signaling disruption has emerged as a potential mechanism underlying neurodegenerative pathology, particularly in Alzheimer's disease (AD). The complex relationship involves:

• ApoER2-DAB1 pathway disruption:

  • Disruption in ApoER2-DAB1 signaling is linked to Tau hyperphosphorylation in humans

  • This pathway normally suppresses GSK3β-mediated Tau phosphorylation

  • When disrupted, it leads to increased pTau formation and neurofibrillary tangles

• DAB1 accumulation patterns:

  • In AD, DAB1 accumulates in swollen dystrophic neurites surrounding ApoE-enriched neuritic plaques

  • DAB1 accumulates in both MAP2-labeled dendrites and NFL-positive axons

  • This accumulation co-localizes with phosphorylated forms of P85α, PSD95, and Tau

• Role as a convergence point:

  • DAB1 serves as a cytoplasmic adaptor for both ApoER2 and amyloid precursor protein (AβPP)

  • ApoER2-DAB1 signaling regulates AβPP cleavage and Aβ synthesis

  • This positions DAB1 as a potential convergence point linking ApoE to both Aβ and pTau pathologies

• Evidence in early stages:

  • DAB1 accumulation has been observed in cases with substantial Aβ plaques but minimal pTau pathology

  • This suggests DAB1 disruption may be an early event in AD pathogenesis

  • These early plaque-associated DAB1 lesions appear before widespread pTau pathology

This mechanistic understanding provides potential therapeutic targets in the ApoER2-DAB1 signaling pathway for neurodegenerative disorders.

What are the most effective experimental approaches to study DAB1 phosphorylation dynamics in primary neuronal cultures?

Studying DAB1 phosphorylation dynamics requires sophisticated experimental approaches:

• Real-time phosphorylation monitoring:

  • Utilize FRET-based biosensors incorporating DAB1 phosphorylation sites

  • Live-cell imaging with phospho-specific antibodies

  • Time-lapse microscopy following Reelin stimulation

• Reelin stimulation protocols:

  • Prepare Reelin-conditioned medium from HEK293 cells expressing Reelin

  • Purify recombinant Reelin for controlled concentration experiments

  • Use phosphatase inhibitors to prevent dephosphorylation during processing

• Quantitative phosphoproteomic analysis:

  • Mass spectrometry to identify multiple phosphorylation sites

  • Phospho-peptide enrichment followed by LC-MS/MS

  • SILAC labeling for comparative phosphorylation analysis

• Kinase inhibitor screens:

  • Target Src family kinases (Src, Fyn, Abl) known to interact with DAB1

  • Use specific inhibitors to dissect phosphorylation pathways

  • Combine with phospho-specific antibodies for Western blot validation

• Mutation strategies:

  • Generate tyrosine-to-phenylalanine mutations at key phosphorylation sites

  • Create phosphomimetic mutations (tyrosine-to-glutamate)

  • Use CRISPR/Cas9 for endogenous DAB1 modification

• Subcellular localization studies:

  • Examine phosphorylated DAB1 trafficking using fluorescent protein fusions

  • Co-localization with cellular compartment markers

  • Super-resolution microscopy for detailed spatial information

These approaches help determine how DAB1 phosphorylation affects neuronal migration, dendritic development, and synaptic plasticity.

How can researchers use DAB1 antibodies to investigate the intersection of Reelin signaling with other neuronal pathways?

DAB1 antibodies serve as valuable tools for investigating pathway crosstalk:

• Co-immunoprecipitation approaches:

  • Use DAB1 antibodies to pull down complexes and identify interacting partners

  • Analyze by mass spectrometry to discover novel DAB1-interacting proteins

  • Verify interactions with reverse co-IP and Western blotting

• Multiplex immunohistochemistry:

  • Combine DAB1 antibodies with markers for:

    • PI3K/Akt pathway components (pP85α Tyr607)

    • Synaptic proteins (PSD95)

    • Cytoskeletal regulators (LIMK1)

    • Tau phosphorylation markers

  • This approach has revealed that DAB1 accumulates together with phosphorylated forms of several signaling partners that stabilize actin, microtubules, and postsynaptic complexes

• Pathway perturbation experiments:

  • Manipulate Reelin signaling while monitoring other pathways:

    • AβPP processing and Aβ synthesis

    • GSK3β activity and Tau phosphorylation

    • Actin cytoskeleton dynamics

  • Use DAB1 antibodies to track changes in complex formation

• Genetic interaction studies:

  • Combine DAB1 knockdown/knockout with manipulation of other pathway components

  • Use conditional and inducible approaches for temporal control

  • Analyze synergistic or antagonistic effects on neuronal phenotypes

• Human pathological sample analysis:

  • Compare DAB1 levels and localization across neurodevelopmental and neurodegenerative conditions

  • Correlate DAB1 changes with other pathway markers

  • Stratify by APOE genotype to identify gene-specific effects

These approaches have revealed important connections between the ApoER2-DAB1 pathway and Alzheimer's disease pathology, including the finding that DAB1 accumulation occurs in both APOE3 homozygotes and APOE2/APOE3 heterozygotes with mild cognitive impairment and Alzheimer's disease, suggesting that ApoER2-DAB1 disruption is a shared mechanism underlying sporadic Alzheimer's disease that may be exacerbated by, but is not dependent on, the APOE4 gene variant .

What are the common challenges in Western blot detection of DAB1 and how can they be resolved?

Western blot detection of DAB1 presents several challenges:

• Variable molecular weight bands:

  • Challenge: DAB1 appears at multiple molecular weights (36-120 kDa)

  • Solution: Include positive controls with known DAB1 expression; expect bands at 45 kDa and 80 kDa with most antibodies

  • Methodology: Use gradient gels (4-15%) to better resolve multiple DAB1 isoforms

• Low signal intensity:

  • Challenge: DAB1 expression may be low in some tissues

  • Solution: Increase protein loading (50-100 μg), extend primary antibody incubation to overnight at 4°C

  • Methodology: Use enhanced chemiluminescence detection systems with longer exposure times

• Background issues:

  • Challenge: Non-specific binding causing high background

  • Solution: Optimize blocking (5% BSA often works better than milk for phospho-epitopes)

  • Methodology: Increase washing time and volume; consider using 0.1% Tween-20 in TBS

• Phosphorylation-specific detection:

  • Challenge: Phospho-epitopes can be lost during sample preparation

  • Solution: Include phosphatase inhibitors in all buffers

  • Methodology: Use fresh samples and avoid repeated freeze-thaw cycles

• Cross-reactivity concerns:

  • Challenge: Some antibodies may cross-react with related proteins

  • Solution: Validate with knockout/knockdown controls

  • Methodology: Pre-absorb antibody with blocking peptide when available

When optimizing, remember that DAB1 antibodies typically work best at 1:1000-1:4000 dilution for Western blotting applications .

How can researchers distinguish between different DAB1 isoforms in their experiments?

DAB1 exists in multiple isoforms, presenting specific research challenges:

• Isoform-specific detection strategies:

  • Use antibodies targeting unique regions of specific isoforms

  • Perform RT-PCR with isoform-specific primers before protein analysis

  • Combine with mass spectrometry to definitively identify isoforms

• Molecular weight differentiation:

  • Use high-resolution SDS-PAGE (10-12% gels) to separate closely migrating isoforms

  • Standard DAB1 isoforms appear at 45 kDa and 80 kDa

  • Larger fusion proteins such as GFP-DAB1 appear at approximately 110 kDa

• Phosphorylation status analysis:

  • Different DAB1 isoforms may show distinct phosphorylation patterns

  • Use phosphatase treatment to identify bands that represent phosphorylated forms

  • Employ phospho-specific antibodies to distinguish activation states

• Subcellular fractionation:

  • Different DAB1 isoforms may localize to different cellular compartments

  • Prepare nuclear, cytoplasmic, membrane, and cytoskeletal fractions

  • Analyze each fraction separately by Western blotting

• Expression pattern analysis:

  • Different DAB1 isoforms may show tissue-specific or developmental stage-specific expression

  • Compare expression across brain regions and developmental timepoints

  • Consider analyzing splicing factor expression that may regulate isoform production

This multi-faceted approach helps researchers accurately identify and characterize the specific DAB1 isoforms relevant to their research questions.

How are DAB1 antibodies being used to investigate the role of ApoER2-DAB1 signaling in Alzheimer's disease mechanisms?

Recent research has employed DAB1 antibodies to reveal novel aspects of Alzheimer's disease pathophysiology:

• Plaque-associated DAB1 accumulation studies:

  • DAB1 antibodies have identified large DAB1 complexes in the molecular layer of the dentate gyrus and CA2 region of the hippocampus in sporadic Alzheimer's disease

  • Similar large DAB1 complexes were observed in three layers (stratum oriens, stratum pyramidale, stratum radiatum) of the prosubiculum-CA1 border region

  • These studies revealed that globular DAB1 primarily accumulates within MAP2-labeled swollen, dystrophic dendrites in the vicinity of ApoE-enriched neuritic plaques

• Early pathology detection:

  • DAB1 antibodies have identified plaque-associated DAB1 accumulation in cases with substantial Aβ plaques but minimal pTau pathology

  • This suggests that DAB1 accumulation may be an early event in Alzheimer's disease pathogenesis that precedes widespread pTau pathology

• Pathway interaction mapping:

  • Multiplex immunohistochemistry combining DAB1 antibodies with markers for P85α, PSD95, and pTau has revealed that DAB1 accumulates together with phosphorylated forms of multiple ApoER2-DAB1-P85α/PI3K signaling partners

  • This approach has provided insights into molecular pathways linking DAB1 accumulation to cytoskeletal instability and synapse loss

• APOE genotype correlation studies:

  • DAB1 antibodies have been used to compare accumulation patterns across different APOE genotypes

  • Findings indicate that extensive DAB1 accumulation occurs in APOE3 homozygotes and APOE2/APOE3 heterozygotes with mild cognitive impairment and Alzheimer's disease

  • This suggests that ApoER2-DAB1 disruption is a shared mechanism underlying sporadic Alzheimer's disease that is not dependent on the APOE4 gene variant

These approaches are revealing DAB1 as a potential convergence point linking ApoE to both Aβ and pTau pathologies, providing new therapeutic targets for Alzheimer's disease intervention.

What are the latest techniques for combining DAB1 antibodies with other markers in multiplex immunohistochemistry studies?

Cutting-edge multiplex immunohistochemistry (MP-IHC) approaches with DAB1 antibodies include:

• Sequential multiplex immunofluorescence:

  • Tyramide signal amplification (TSA) allows multiple primary antibodies from the same species

  • Sequential staining, imaging, and quenching cycles

  • Has revealed that DAB1, pP85α Tyr607, and pPSD95 Thr19 accumulate together with pTau in swollen dystrophic neurites surrounding ApoE-enriched neuritic plaques

• Mass cytometry imaging:

  • Metal-conjugated antibodies detected by mass spectrometry

  • Allows simultaneous detection of >40 markers

  • Particularly useful for mapping complex signaling networks involving DAB1

• Spatial transcriptomics integration:

  • Combine DAB1 protein detection with RNA analysis

  • Correlate protein accumulation with gene expression changes

  • Map spatial relationships between DAB1 and expression of related pathway components

• Super-resolution microscopy approaches:

  • STED, STORM, or PALM microscopy for nanoscale resolution

  • Reveals detailed subcellular localization of DAB1 in relation to other markers

  • Essential for examining DAB1 in small neuronal compartments like dendritic spines

• Clearing techniques with whole-organ immunolabeling:

  • CLARITY, iDISCO, or CUBIC tissue clearing

  • Allows 3D visualization of DAB1 distribution throughout brain regions

  • Particularly valuable for tracing neuronal connectivity

These techniques have enabled researchers to discover that DAB1 accumulates in both MAP2-labeled dendrites and NFL-labeled axons in Alzheimer's disease, with layer-specific differences in accumulation patterns. For example, in the stratum radiatum layer, DAB1 accumulates primarily within NFL-positive dystrophic axons, while in the stratum pyramidale layer, it accumulates in MAP2-labeled dendrites .