DOCK7 Antibody, FITC conjugated

What is DOCK7 and what are its primary functions in neuronal development?

DOCK7 (dedicator of cytokinesis 7) is a 2,140 amino acid protein that functions as a guanine nucleotide exchange factor (GEF) specifically activating Rac1 and Rac3 by catalyzing the exchange of bound GDP for free GTP. It localizes to developing axons and contains one DHR-1 domain and one DHR-2 domain . DOCK7 plays a critical role in axon formation and neuronal polarization, as evidenced by its expression primarily in neuronal cells . Multiple isoforms exist due to alternative splicing events, and the protein is encoded by gene ID 85440 .

Methodologically, when investigating DOCK7's neuronal functions, researchers should consider dual immunostaining approaches that visualize both DOCK7 and cytoskeletal markers to examine their spatial relationships during different developmental stages.

What applications are recommended for DOCK7 antibody, FITC conjugated?

The DOCK7 antibody, FITC conjugated, is specifically designed for immunofluorescence applications including:

This FITC-conjugated format eliminates the need for secondary antibodies, reducing experimental variables and potential cross-reactivity issues in multi-labeling experiments .

What species reactivity can be expected with DOCK7 antibody, FITC conjugated?

The reactivity profile for DOCK7 antibody, FITC conjugated includes:

When planning experiments with predicted reactive species, preliminary validation experiments should be performed to confirm specificity before proceeding with comprehensive studies .

What are the optimal storage and handling conditions for maintaining antibody integrity?

For maximum stability and activity retention of DOCK7 antibody, FITC conjugated:

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• The storage buffer (0.01M TBS pH 7.4 with 1% BSA, 0.03% Proclin300, and 50% Glycerol) is designed to maintain antibody stability

• When handling, minimize exposure to light during all experimental procedures

Researchers should monitor fluorescence intensity across experiments as a quality control measure for antibody performance.

How can DOCK7 antibody be used to investigate directional cell migration mechanisms?

DOCK7 plays a crucial role in directional cell migration, particularly in cancer contexts. Research shows that DOCK7 inhibition results in cells migrating more randomly with reduced directionality and velocity .

To investigate this using DOCK7 antibody, FITC conjugated:

• Experimental design:

  • Implement wound healing assays with time-lapse imaging

  • Compare control cells versus DOCK7-knockdown cells

  • Track individual cell trajectories to calculate directionality index (net distance/total distance)

• Immunofluorescence analysis:

  • Fix cells at multiple timepoints (0h, 6h, 12h, 24h) during migration

  • Stain with DOCK7 antibody, FITC conjugated (1:50 dilution)

  • Co-stain with focal adhesion markers (paxillin, vinculin, FAK)

• Quantitative assessment:

  • Measure directionality index = net distance/total distance traveled

  • Calculate migration velocity

  • Analyze DOCK7 localization relative to the leading edge

This methodological approach allows visualization of how DOCK7 influences the orchestration of migration machinery components required for directional persistence .

What controls are essential when using DOCK7 antibody, FITC conjugated in immunofluorescence studies?

When performing immunofluorescence with DOCK7 antibody, FITC conjugated, the following controls are essential:

These controls help distinguish genuine DOCK7 staining from technical artifacts, especially important when investigating novel functions or cellular localizations.

How can DOCK7 antibody be used to investigate its role in cytoskeletal reorganization?

DOCK7's function in cytoskeletal reorganization can be studied using DOCK7 antibody, FITC conjugated through several methodological approaches:

• Cytoskeletal co-localization studies:

  • Triple staining with DOCK7 (FITC, green), actin (phalloidin, red), and microtubules (α-tubulin, far-red)

  • Confocal microscopy to analyze spatial relationships at the leading edge of migrating cells

• Focal adhesion dynamics:

  • Co-stain for DOCK7 and focal adhesion markers (paxillin, vinculin, FAK)

  • Quantify focal adhesion size, number, and distribution in control versus DOCK7-depleted cells

Research has shown that DOCK7 influences:

• Formation of dense actin bundles at the leading edge

• Orientation of microtubules aligned with migration direction

• Assembly and turnover of focal adhesions

• Phosphorylation of focal adhesion proteins (FAK Tyr397, Paxillin)

These methodological approaches can reveal how DOCK7 coordinates cytoskeletal components required for migration and invasion.

What approaches can be used to investigate DOCK7's role in 3D invasion models?

To study DOCK7's function in 3D invasion using DOCK7 antibody, FITC conjugated:

• 3D matrix invasion assay setup:

  • Prepare collagen and fibronectin-rich plugs as described in literature

  • Seed control and DOCK7-knockdown cells on matrices

  • Perform time-lapse imaging over 3 days

• Quantitative analysis:

  • Measure invasion depth (using 45μm as threshold distance)

  • Calculate percentage of invasive cells

  • Compare invasion strategies between control and DOCK7-inhibited cells

• Immunofluorescence in 3D:

  • Fix 3D cultures with 4% PFA for extended time (30-60 min)

  • Permeabilize thoroughly (0.5% Triton X-100, 30 min)

  • Stain with DOCK7 antibody, FITC conjugated (1:50 dilution, overnight at 4°C)

  • Counter-stain for invasion markers

Research shows DOCK7 inhibition reduces both invasion depth and the proportion of invasive cells in 3D matrices, with DOCK7-depleted cells traveling approximately 2.5 times less deep compared to control cells .

How can DOCK7 antibody be used to correlate in vitro findings with in vivo metastatic potential?

To bridge in vitro observations with in vivo metastatic capacity:

• In vitro characterization:

  • Establish stable cell lines (control, DOCK7-knockdown)

  • Confirm knockdown efficiency using DOCK7 antibody in immunofluorescence

  • Validate migration/invasion phenotypes in 2D and 3D assays

• In vivo metastasis model:

  • Inject characterized cells into mouse tail vein

  • Monitor lung colonization (6 weeks post-injection)

  • Analyze lung tissue sections with H&E staining and immunofluorescence

Research demonstrates that DOCK7 inhibition significantly reduces pulmonary metastatic potential, with DOCK7-depleted cells forming approximately 1.5-fold fewer lung nodules compared to control cells .

This translational approach connects molecular mechanisms to physiological outcomes, highlighting DOCK7's clinical relevance in cancer progression.

What are the recommended fixation and permeabilization protocols for optimal DOCK7 antibody performance?

For optimal results with DOCK7 antibody, FITC conjugated:

When working with different sample types:

• For neuronal cells: Consider gentler permeabilization (0.1% Triton X-100, 5 min)

• For tissue sections: Optimize antigen retrieval (citrate buffer, pH 6.0)

• For 3D cultures: Extend all incubation times by 2-3×

These parameters should be optimized based on specific experimental conditions to achieve the best signal-to-noise ratio.

How can researchers address weak or non-specific signals when using DOCK7 antibody?

When troubleshooting suboptimal staining patterns:

For weak signals:

• Reduce antibody dilution (try 1:50 instead of 1:200)

• Extend primary antibody incubation (overnight at 4°C)

• Optimize fixation conditions (test both PFA and methanol)

• Try antigen retrieval methods for tissues or strongly fixed samples

For non-specific binding:

• Increase blocking time (2 hours minimum)

• Add 0.1% Tween-20 to wash buffers

• Filter antibody solution through 0.22μm filter

• Pre-absorb antibody with non-expressing cell lysate

Systematic optimization of these parameters will help achieve specific DOCK7 detection while minimizing background.

How should experiments be designed to investigate DOCK7's interaction with Rac GTPases?

To study DOCK7-Rac interactions using DOCK7 antibody, FITC conjugated:

• Co-localization analysis:

  • Stain cells with DOCK7 antibody, FITC conjugated (green channel)

  • Counter-stain with Rac1/Rac3 antibodies (red channel)

  • Analyze co-localization at the leading edge of migrating cells

• Functional studies:

  • Compare Rac activation (using GST-PAK-CRIB pulldown) in control vs. DOCK7-depleted cells

  • Rescue experiments with constitutively active Rac1/Rac3

• Experimental conditions to include:

  • Resting cells vs. stimulated cells (growth factors)

  • Wild-type vs. dominant-negative Rac mutants

  • Various time points after stimulation

This experimental design enables both visualization and functional analysis of DOCK7's role as a GEF for Rac GTPases, correlating its localization with activation zones in migrating cells.

What methodological approaches can distinguish between DOCK7's role in axonal development versus cancer cell migration?

To differentiate DOCK7's tissue-specific functions:

• Comparative cellular models:

  • Neuronal cells (primary neurons or neuronal lines)

  • Cancer cell lines (with varying metastatic potential)

  • Normal epithelial cells (as controls)

• Phenotypic analysis:

  • Neurons: Assess axon specification, length, branching

  • Cancer cells: Measure migration directionality, invasion depth

  • Both: Analyze cytoskeletal organization using DOCK7 antibody, FITC conjugated

• Molecular pathway analysis:

  • Investigate downstream effectors in each context

  • Perform rescue experiments with tissue-specific factors

This comparative approach can identify both conserved and context-specific functions of DOCK7, potentially revealing therapeutic opportunities in pathological settings without disrupting normal neuronal functions.

How should researchers quantify DOCK7 localization patterns in different cellular compartments?

For rigorous quantification of DOCK7 localization using DOCK7 antibody, FITC conjugated:

• Image acquisition parameters:

  • Capture z-stack confocal images (0.5-1μm intervals)

  • Maintain consistent laser power and detector settings across samples

  • Include co-staining with compartment markers

• Quantification methodology:

  • Define regions of interest (ROIs) for specific compartments

  • Measure mean fluorescence intensity within each ROI

  • Calculate relative distribution (% of total cellular signal)

• Statistical analysis:

  • Compare at least 30-50 cells per condition

  • Apply appropriate statistical tests (ANOVA with post-hoc for multiple comparisons)

  • Present data as box plots showing distribution of values

This standardized approach enables objective comparison of DOCK7 localization between different experimental conditions, cell types, or disease states.

What considerations are important when interpreting contradictory findings about DOCK7 function?

When faced with apparently contradictory results:

• Methodological reconciliation:

  • Compare antibody epitopes (DOCK7 antibody, FITC conjugated targets region 1401-1500/2140)

  • Evaluate cell type differences (neuronal vs. epithelial vs. cancer cells)

  • Assess knockdown/knockout strategies (acute vs. chronic depletion)

• Context-dependent interpretation:

  • DOCK7 may interact with different effectors in different cell types

  • Post-translational modifications might alter function in specific contexts

  • Compensatory mechanisms may emerge in certain experimental systems

• Reconciliation strategies:

  • Perform rescue experiments with full-length and domain-specific constructs

  • Test activity in defined in vitro systems

  • Use complementary approaches (biochemical, genetic, imaging)

This systematic analysis of seemingly contradictory findings can often reveal nuanced regulatory mechanisms and context-specific functions of DOCK7.