DABB1 Antibody

What are the primary applications for DAB1 antibodies in neuroscience research?

DAB1 antibodies are versatile tools for investigating Reelin-mediated signaling pathways in brain development and function. The most validated applications include:

• Western Blotting (WB): Most DAB1 antibodies are validated for WB, typically detecting bands at approximately 64-80 kDa depending on the isoform and post-translational modifications .

• Immunohistochemistry on paraffin sections (IHC-P): Useful for examining DAB1 expression patterns in different brain regions .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Enables visualization of DAB1 subcellular localization .

• Immunoprecipitation (IP): Allows isolation of DAB1 protein complexes to study interaction partners .

Methodology note: When performing immunofluorescence, optimal dilutions range from 1/500 to 1/1000 for most commercial antibodies .

How can I validate the specificity of a DAB1 antibody?

Methodological approach for DAB1 antibody validation:

• Use appropriate controls:

  • Positive control: Brain lysates from wild-type mice, particularly from P14 brain tissue when DAB1 expression is high .

  • Negative control: Brain tissue from DAB1-deficient mice (scrambler mutants or Dab1 KO) .

• Verify expected band size:

  • The predicted band size for DAB1 is approximately 64 kDa .

  • In WB applications, observed band sizes may range from 67-80 kDa depending on post-translational modifications .

  • GFP-DAB1 fusion proteins appear at approximately 110 kDa .

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide to confirm specificity .

The research by Trotter et al. (2013) demonstrated specificity by comparing staining in wild-type mice versus scrambler (Scm) mutants, showing prominent DAB1 signal throughout the forebrain of wild-type mice, but not in Scm mutants .

What are the differences between phospho-specific and total DAB1 antibodies?

Methodological insight: Research by Trotter et al. (2013) and Arnaud et al. (2003) indicates that the phosphorylation of DAB1 at tyrosine residues is crucial for its function in Reelin signaling and for downregulation of DAB1 levels through ubiquitin-mediated proteolysis .

How can DAB1 antibodies be used to investigate Reelin signaling in neuronal development?

Methodological approach for studying Reelin-DAB1 signaling pathway:

• Investigating phosphorylation dynamics:

  • Use phospho-specific antibodies (p-Y220, p-Y232) to monitor activation of DAB1 following Reelin stimulation .

  • Compare phosphorylation levels across developmental stages (P7, P14, P28, P56) to track changing signaling dynamics .

• Analyzing protein interactions:

  • Use co-immunoprecipitation with DAB1 antibodies to identify binding partners like Crk, CrkL, and Src family kinases (SFKs) .

  • Examine how phosphorylation affects the formation of signaling complexes.

• Subcellular localization studies:

  • Triple immunofluorescence labeling with DAB1, PSD-95 (postsynaptic marker), and synaptophysin (presynaptic marker) can reveal synaptic localization of DAB1 .

  • Focus on specific regions like the stratum radiatum of hippocampal area CA1 to examine dendritic localization.

Research by Arnaud et al. (2003) demonstrated that different phosphorylation sites on DAB1 serve distinct functions: two tyrosines are required for Reelin to stimulate DAB1 phosphorylation at other sites and to activate Akt, while two other tyrosines are required to stimulate a Crk/CrkL-C3G pathway .

What roles do different DAB1 phosphorylation sites play in neuronal signaling pathways?

DAB1 contains multiple tyrosine phosphorylation sites with distinct functions in signaling:

Research by Arnaud et al. (2003) using complementation analysis demonstrated that the different phosphorylation sites have independent functions that are needed together for normal neuronal lamination .

How does DAB1 gene dosage affect brain development and function?

Recent research on DAB1 gene dosage effects reveals:

• Regional sensitivity differences:

  • Neocortex: DAB1 is haplosufficient for neuronal migration but haploinsufficient for control of layer 1 thickness .

  • Hippocampus: Migration of a subpopulation of pyramidal neurons is sensitive to DAB1 gene haploinsufficiency .

• Signaling pathway dose-dependence:

  • The PI3K/Akt pathway shows reduced basal activity (55.7% of wild-type in cortex) in DAB1-deficient adult mice .

  • ERK1/2 signaling is similarly affected (57.3% of wild-type in cortex) .

• Functional consequences:

  • Adult forebrain-specific DAB1 deletion impairs synaptic plasticity and hippocampal-dependent learning .

  • These abnormalities are associated with reduced dendritic spine size and defects in basal and plasticity-induced signaling .

Methodological approach: Study by Trotter et al. (2013) used conditional knockout mice to overcome limitations of constitutive knockouts, enabling investigation of DAB1's role specifically in the adult forebrain .

How can I optimize immunohistochemical detection of DAB1 in brain tissue?

Methodological recommendations for optimal DAB1 immunostaining:

• Fixation and tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissue for consistent results .

  • For immunofluorescence on frozen sections, brief fixation with 4% paraformaldehyde preserves epitope accessibility.

• Antigen retrieval:

  • Heat-mediated antigen retrieval improves detection, particularly for phospho-epitopes.

• Antibody dilution and incubation:

  • For IHC-P: Optimal dilutions range from 1/100 to 1/500 .

  • For ICC/IF: Dilutions of 1/500 to 1/1000 work well for most antibodies .

  • Extended incubation at 4°C overnight often improves signal-to-noise ratio.

• Controls and validation:

  • Use brain sections from DAB1-deficient mice as negative controls .

  • For phospho-specific antibodies, treatment with lambda phosphatase can confirm phospho-specificity.

Trotter et al. (2013) successfully used the DAB1 B3 antibody at 1/500 dilution for immunofluorescence, showing widespread DAB1 expression in the forebrain with particularly intense signal in cell bodies of the pyramidal layer and in the neuropil of the stratum lacunosum moleculare .

Why might I observe multiple bands when probing for DAB1 in Western blots?

Multiple bands in DAB1 Western blots may represent:

• Different DAB1 isoforms:

  • The canonical band size is approximately 64 kDa .

  • Alternative splicing produces multiple isoforms with different molecular weights.

  • p80 is a common splice form used in experimental systems .

• Post-translational modifications:

  • Phosphorylated forms may show slight shifts in mobility.

  • Ubiquitination can produce higher molecular weight species, especially when examining DAB1 turnover.

• Degradation products:

  • C-terminal antibodies may detect lower molecular weight fragments.

• Fusion proteins in experimental systems:

  • GFP-DAB1 fusion proteins appear at approximately 110 kDa .

Methodological approach to verify bands:

• Use multiple antibodies targeting different epitopes of DAB1.

• Include positive controls (e.g., transfected cells expressing DAB1) .

• For phospho-specific antibodies, include phosphatase-treated samples as controls.

How can DAB1 antibodies contribute to understanding neurodevelopmental disorders?

DAB1 antibodies can advance research on neurodevelopmental disorders through:

• Investigation of Reelin pathway disruptions:

  • Attenuation of Reelin signaling has been associated with schizophrenia, bipolar disorder, autism spectrum disorder, and Alzheimer's disease .

  • Phospho-specific DAB1 antibodies can assess pathway integrity in patient-derived samples or disease models.

• Molecular characterization of circuit formation:

  • DAB1 is critical for synaptic plasticity and associative learning .

  • Antibodies can help map DAB1 expression and phosphorylation in specific neural circuits affected in disorders.

• Therapeutic target validation:

  • Monitoring DAB1 phosphorylation as a readout for compounds targeting Reelin pathway components.

Methodological approach: Trotter et al. (2013) demonstrated that adult forebrain-specific and excitatory neuron-specific conditional knockout of DAB1 leads to defects in synaptic function and hippocampal-dependent learning, providing a valuable model system for investigating DAB1's role in cognitive disorders .

What approaches can be used to study the interaction between DAB1 and other signaling proteins?

Advanced methodological approaches include:

• Proximity ligation assays (PLA):

  • Combine DAB1 antibodies with antibodies against potential interaction partners.

  • PLA provides spatial resolution of protein interactions in situ.

• Co-immunoprecipitation followed by mass spectrometry:

  • Use validated DAB1 antibodies for pull-down experiments.

  • Identify novel interaction partners and how they change with Reelin stimulation.

• FRET/FLIM microscopy:

  • Combine fluorescently labeled antibodies to measure protein proximity in living cells.

Research by Arnaud et al. (2003) used co-immunoprecipitation to demonstrate that Reelin stimulates the binding of Fyn to normal DAB1 or mutant DAB1cd proteins, but not to mutant DAB1ab, revealing important insights about the structural requirements for DAB1-SFK interactions .