DOCK3 Antibody

What is DOCK3 and why is it significant in neuroscience research?

DOCK3 (Dedicator of Cytokinesis 3) is an atypical guanine nucleotide exchange factor (GEF) that plays critical roles in neuronal development and function. It is primarily expressed in the brain, spinal cord, and retina, being the only DOCK protein that shows almost exclusive expression in the central nervous system (CNS) . Research significance includes:

• DOCK3 activates the small GTPase Rac1, influencing cytoskeletal remodeling

• It stimulates axonal outgrowth and regeneration after injury

• DOCK3 is associated with Alzheimer's disease tangles and affects amyloid precursor protein metabolism

• Deletion of DOCK3 in mice results in axon degeneration and sensorimotor impairments

• Loss-of-function DOCK3 variants in humans cause developmental delay and hypotonia

What are the optimal epitope regions to target when selecting a DOCK3 antibody?

When selecting DOCK3 antibodies, consider targeting functionally important domains:

Choose antibodies targeting conserved epitopes for cross-species studies, while those recognizing species-specific regions may offer higher specificity for single-species experiments .

How should I validate DOCK3 antibody specificity before experimental use?

Thorough validation is essential for reliable results:

• Genetic controls: Test in DOCK3 knockout/knockdown models (conditional knockout mice with Cre-mediated excision of exons 8 and 9 have been validated)

• Phosphatase treatment: Verify phosphorylated forms by treating samples with phosphatase and observing mobility shifts in Western blots

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Cross-reactivity assessment: Test against human, mouse, and rat samples if planning cross-species studies

• Multiple application validation: Confirm specificity across different applications (WB, IHC, IF)

What are the recommended protocols for DOCK3 detection in Western blotting?

For optimal Western blot detection of DOCK3:

Sample preparation:

• Use brain tissue, SH-SY5Y cells, or specialized neuronal cultures

• Include phosphatase inhibitors to preserve phosphorylated forms

Protocol parameters:

• Expected molecular weight: ~233 kDa (full-length DOCK3)

• Recommended dilutions: 1:2000-1:16000 for most commercial antibodies

• Buffer system: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Positive controls: Mouse brain tissue, human brain tissue, SH-SY5Y cells, rat brain tissue

Detection considerations:

• Be aware of phosphorylated forms appearing as upper mobility bands

• Use phosphatase treatment to confirm phosphorylated species

What are the optimal conditions for immunohistochemical detection of DOCK3 in neural tissues?

For successful immunohistochemical detection:

Tissue preparation:

• Use immersion-fixed paraffin-embedded sections

• For Alzheimer's research, human cortex sections show strong localization to neurofibrillary tangles

Antigen retrieval methods:

• Primary recommendation: TE buffer (pH 9.0)

• Alternative method: Citrate buffer (pH 6.0)

Staining protocol:

• Recommended dilutions: 1:50-1:500 for most commercial antibodies

• For visualization: Anti-Sheep HRP-DAB staining system works effectively for sheep-derived antibodies

• Counterstain with hematoxylin for structural context

Specialized applications:

• For retinal ganglion cells (RGCs), DOCK3 concentrates in growth cones

• Following BDNF treatment, observe DOCK3 rearrangement to cell periphery

How can I optimize co-immunoprecipitation experiments to study DOCK3 protein interactions?

For successful co-IP studies of DOCK3 interactions:

Validated protocols:

• His-tag pull-down assays effectively demonstrate DOCK3 binding to WAVE proteins

• Anti-FLAG M2 magnetic beads for FLAG-tagged fusion proteins

• μMACS HA magnetic bead isolation kit for HA-tagged constructs

Buffer conditions:

• For GSK-3β interactions: Use buffers that preserve phosphorylation states

• For WAVE protein interactions: Standard IP buffers are sufficient

Controls and validation:

• Include phosphatase treatments to assess phosphorylation-dependent interactions

• Use truncated mutants to map interaction domains:

  • DOCK3 mutants lacking specific regions (e.g., 1-500, 1-1540, 1-1777, ΔDHR-1, ΔDHR-2)

  • Test WAVE protein mutants lacking the WAVE-homology domain (WHD)

How can DOCK3 antibodies be used to investigate the mechanisms of axonal regeneration?

DOCK3 plays crucial roles in axonal regeneration through multiple mechanisms:

Experimental approaches:

• Subcellular localization studies:

  • Use immunofluorescence to track DOCK3 redistribution after BDNF treatment

  • Observe DOCK3 concentration in growth cones and translocation to cell periphery

• Protein-protein interaction analysis:

  • Investigate DOCK3-WAVE complexes using co-IP and immunofluorescence

  • Study DOCK3-GSK-3β interactions and phosphorylation states

• Functional studies:

  • Compare DOCK3 overexpression effects on axonal outgrowth in transgenic models

  • Evaluate DOCK3 interaction with BDNF-TrkB signaling pathways

  • Assess Rac1 activation downstream of DOCK3 using pull-down assays

Key findings to validate:

• DOCK3 overexpression increases BDNF-mediated axonal outgrowth

• DOCK3 siRNA inhibits BDNF effects on axon extension

• Membrane-targeted forms of DOCK3 (F-DOCK3) show enhanced activity

What methods should I use to study DOCK3's role in Alzheimer's disease pathology?

DOCK3 has significant implications in Alzheimer's disease research:

Experimental approaches:

• Immunohistochemical analysis:

  • Use validated DOCK3 antibodies to examine localization in AD brain samples

  • Compare expression in soluble fractions of AD brain vs. age-matched controls

  • Examine co-localization with neurofibrillary tangles

• Functional studies:

  • Investigate DOCK3's effect on APP secretion and degradation

  • Examine interaction with β-amyloid processing pathways

• Molecular mapping:

  • Use domain-specific antibodies to identify functional regions involved in AD pathology

  • Examine phosphorylation states in AD vs. normal tissue

Technical considerations:

• For human AD tissue: Use sheep anti-human DOCK3 antibody at 3 μg/mL (overnight at 4°C)

• For visualization: Anti-Sheep HRP-DAB staining system with hematoxylin counterstain

• Focus on cortical regions for strongest pathology associations

How can I use DOCK3 antibodies to analyze its phosphorylation state and regulatory mechanisms?

DOCK3 activity is regulated by phosphorylation, which affects its interactions:

Detection methods:

• Gel mobility shift assays:

  • Phosphorylated DOCK3 appears as an upper mobility band in SDS-PAGE

  • Membrane-targeted F-DOCK3 shows enhanced phosphorylation (26±6% of total DOCK3)

  • Wild-type DOCK3 exhibits minimal phosphorylation (2±1% of total)

• Phospho-specific approaches:

  • Treat samples with phosphatase to confirm phosphorylated species

  • Use GSK-3β phosphorylation as a readout of DOCK3 activity

• Functional correlation:

  • Analyze binding affinity differences between phosphorylated and non-phosphorylated forms:

    • Non-phosphorylated DOCK3 binds WAVE1 more effectively

    • Phosphorylation may trigger DOCK3/WAVE1 complex dissociation

Experimental design tips:

• Include phosphatase inhibitors in lysate preparation

• For interaction studies, separate phosphorylated forms via gel migration

• Compare wild-type vs. F-DOCK3 (membrane-targeted) for phosphorylation state analysis

How do I resolve weak or non-specific signals when using DOCK3 antibodies?

When encountering signal problems with DOCK3 antibodies:

For weak signals:

• Antigen retrieval optimization:

  • Primary method: TE buffer (pH 9.0)

  • Alternative: Citrate buffer (pH 6.0)

  • Extend retrieval time for fixed tissues

• Antibody concentration adjustment:

  • For WB: Test range from 1:2000 to 1:16000

  • For IHC: Test range from 1:50 to 1:500

  • Incubate longer at 4°C (overnight) for challenging samples

• Sample enrichment:

  • Focus on CNS tissues with high endogenous expression

  • Consider subcellular fractionation to concentrate DOCK3

For non-specific signals:

• Blocking optimization:

  • Use 0.1% BSA in buffer systems for reduced background

  • Extend blocking time to minimize non-specific binding

• Antibody validation:

  • Test in DOCK3 knockout/knockdown models

  • Use peptide competition to confirm specific binding

• Application-specific adjustments:

  • For IHC: Dilute antibody further and extend incubation time

  • For WB: Use longer wash cycles to reduce background

What controls should I include when studying DOCK3 in transgenic or knockout models?

When working with genetic models:

Essential controls:

• Genetic validation:

  • Verify Dock3 gene modification by PCR using primers:

    • F: 5'-GAGATGCTGATTTCACTGTCTAGC-3'

    • R: 5'-CTCTTATCACTGGCTGAAACTACA-3'

  • Confirm Cre recombinase expression with:

    • F: 5'-GAACGCACTGATTTCGACCA-3'

    • R: 5'-GCTAACCAGCGTTTTCGTTC-3'

• Protein expression controls:

  • Wild-type tissue/cells as positive control

  • Complete knockout as negative control

  • Heterozygous samples for dose-response validation

• Functional validation:

  • Test Rac1 activation levels in knockout vs. wild-type

  • Assess axonal growth phenotypes in neuronal cultures

  • Evaluate interaction with known binding partners (WAVE, GSK-3β)

Experimental design considerations:

• Use conditional knockouts for tissue-specific studies

• Include multiple antibodies targeting different DOCK3 epitopes

• Validate phenotypes with rescue experiments using wild-type DOCK3

How do low-molecular-weight compounds that modulate DOCK3 activity interact with antibody-based detection methods?

Recent advances have identified compounds modulating DOCK3-Elmo1 interactions:

Methodological considerations:

• Compound screening approaches:

  • Luciferase complementation assays monitoring DOCK3 intramolecular interactions

  • Assessment of compounds affecting SH3-DHR2 domain interactions

• Validation with antibody-based techniques:

  • Use DOCK3 antibodies to verify protein expression levels remain consistent

  • Employ co-IP to confirm compound effects on protein-protein interactions

  • Apply immunofluorescence to observe subcellular localization changes

• Potential interference issues:

  • Some compounds may alter epitope accessibility

  • Consider testing multiple antibodies targeting different regions

  • Include compound-free controls in all experiments

Research applications:

• Combine compound treatment with immunostaining to assess effects on axon regeneration

• Use antibody-based assays to evaluate neuroprotective effects in optic nerve injury models

• Monitor DOCK3-Elmo1-Rac1 pathway activation with appropriate antibodies

What are the best methodological approaches for studying DOCK3's role in skeletal muscle biology?

Although primarily known for CNS functions, DOCK3 also plays roles in muscle:

Experimental approaches:

• Expression analysis in muscle tissues:

  • Use validated DOCK3 antibodies for Western blot (1:2000-1:16000 dilution)

  • Apply immunohistochemistry for localization studies (1:50-1:500 dilution)

• Functional studies in muscular dystrophy models:

  • DOCK3 is upregulated in Duchenne muscular dystrophy (DMD)

  • Examine skeletal muscles from DMD patients and dystrophic mice

  • Compare with conditional DOCK3 knockout mouse models

• Interaction analysis:

  • Study DOCK3-SORBS1 binding using yeast two-hybrid systems

  • Verify interactions with co-IP using anti-FLAG and anti-HA systems

Technical considerations:

• For muscle tissue: Use specific extraction protocols to preserve membrane-associated fractions

• Include phosphatase inhibitors to maintain phosphorylation states

• Consider skeletal-muscle-specific conditional knockout models (Human Skeletal-Actin-MerCreMer)

What methodological approaches are most effective for studying DOCK3's role in neurodevelopmental disorders?

DOCK3 variants are associated with developmental delay and hypotonia:

Research strategies:

• Patient-derived samples:

  • Apply DOCK3 antibodies to analyze expression in available tissues

  • Compare subcellular localization between patient and control samples

  • Assess phosphorylation states and protein interactions

• Functional assays:

  • Use primary neuronal cultures from genetic models

  • Assess axonal development with quantitative immunofluorescence

  • Monitor DOCK3-dependent Rac1 activation with pull-down assays

• Molecular characterization:

  • Map domain-specific functions using truncation mutants

  • Examine effects of patient-specific mutations on:

    • WAVE protein interactions

    • GSK-3β signaling

    • Membrane localization and phosphorylation

Experimental design considerations:

• Include age-matched controls for developmental studies

• Use multiple antibodies targeting different domains to assess variant effects

• Consider developmental timepoints when designing experiments