DOCK3 Antibody, FITC conjugated

What is DOCK3 and why is it important in research?

DOCK3 (Dedicator of Cytokinesis 3, also known as MOCA or Modifier of Cell Adhesion) is a guanine nucleotide exchange factor (GEF) protein that activates the small GTPase Rac1. DOCK3 plays critical roles in multiple cellular processes:

• Essential for normal muscle function and regeneration

• Regulates glucose metabolism through interaction with SORBS1

• Controls myoblast fusion and differentiation

• Promotes axonal outgrowth in neurons by inactivating GSK-3β

• Modulates cytoskeletal dynamics through WAVE complex interaction

DOCK3 has emerged as a key regulator in muscular dystrophy pathogenesis, with expression levels strongly linked to dystrophic pathologies in zebrafish and mouse models . Recent studies demonstrate that DOCK3 is a dosage-sensitive modulator of skeletal muscle health, making it an important research target .

What is FITC conjugation and how does it enhance antibody functionality?

Fluorescein isothiocyanate (FITC) is a reactive fluorescent dye that covalently binds to proteins at primary amine groups, particularly lysine residues. The conjugation process creates antibodies that emit green fluorescence (emission ~515-524 nm) when excited at ~495 nm, allowing direct visualization without secondary detection reagents.

Key characteristics of FITC conjugation:

• Optimal conjugation occurs at pH 9.5 with high protein concentration (25 mg/ml)

• Maximum labeling can be achieved in 30-60 minutes at room temperature

• The molecular fluorescein/protein (F/P) ratio determines conjugate quality and brightness

• Excitation/emission maxima wavelengths are typically 495 nm/515-524 nm

FITC conjugation enables direct detection of target proteins in applications like immunofluorescence microscopy and flow cytometry without requiring secondary antibody steps.

What are the recommended applications for DOCK3-FITC antibodies?

DOCK3-FITC antibodies are versatile research tools applicable to multiple experimental methods:

DOCK3-FITC antibodies are particularly valuable for investigating:

• Subcellular localization at the plasma membrane

• Co-localization with interaction partners like GSK-3β

• Changes in expression during myoblast differentiation

• Alterations in dystrophic muscle samples

What are the optimal storage conditions for DOCK3-FITC antibodies?

Proper storage is critical for maintaining FITC-conjugated antibody activity and fluorescence:

• Temperature: Store at 2-8°C for short-term use; -20°C for long-term storage

• Buffer composition: PBS with stabilizers (0.09% sodium azide and 0.5% BSA or 50% glycerol)

• Light protection: Essential to prevent photobleaching of the FITC fluorophore

• Aliquoting: Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

• Shelf life: Typically stable for one year after shipment when properly stored

Improper storage can lead to antibody degradation, loss of specificity, and decreased fluorescence intensity. Always validate antibody performance after extended storage periods.

How can researchers validate the specificity of DOCK3-FITC antibodies?

Rigorous validation ensures experimental reliability and reproducibility:

• Western blot analysis confirming a single band at the expected molecular weight (230-240 kDa)

• Testing on DOCK3 knockout samples as negative controls

• Comparison with non-conjugated DOCK3 antibodies targeting the same epitope

• Cross-reactivity testing with other DOCK family members (less than 3% cross-reactivity with DOCK1, DOCK2, and DOCK5 is typical)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Correlation of staining patterns with known DOCK3 expression profiles in tissues

The Human DOCK3 Antibody from R&D Systems, for example, demonstrates less than 3% cross-reactivity with recombinant human DOCK1, DOCK2, and DOCK5 in direct ELISAs .

What controls should be included in DOCK3-FITC antibody experiments?

Proper controls are essential for accurate data interpretation:

For DOCK3 knockout controls, researchers can use tissues from DOCK3 mKO (muscle-specific knockout) or global KO mice that have been characterized in recent studies .

How can DOCK3-FITC antibodies be used to study DOCK3-GSK-3β interaction?

DOCK3 forms a complex with GSK-3β at the plasma membrane, leading to GSK-3β phosphorylation and inactivation, which affects downstream targets like CRMP-2 . To investigate this interaction:

• Use co-immunofluorescence with DOCK3-FITC and anti-phosphorylated-GSK-3β antibodies

• Focus on the DOCK3-GSK-3β binding domain (amino acids 1628-1777 of DOCK3)

• Compare staining patterns of wild-type DOCK3 versus ΔGSK DOCK3 (deletion of residues 1628-1777)

• Employ F-DOCK3 (farnesylated DOCK3) as a positive control for membrane localization

• Quantify co-localization coefficients between DOCK3 and phosphorylated GSK-3β

• Monitor changes in CRMP-2 phosphorylation status as a functional readout

This experimental approach can reveal how DOCK3 regulates microtubule assembly through GSK-3β inactivation and subsequent CRMP-2 dephosphorylation, influencing axonal outgrowth and potentially muscle cell function .

What methodologies can researchers use to investigate DOCK3's role in myoblast fusion?

DOCK3 significantly impacts myoblast fusion and differentiation. To study this process:

• Compare fusion index between control and DOCK3 knockdown myoblasts using immunofluorescence with DOCK3-FITC and myosin heavy chain (MyHC) antibodies

• Quantify myogenic differentiation by calculating the percentage of MyHC-positive cells

• Track DOCK3 localization during different stages of myoblast fusion

• Analyze changes in actin dynamics through co-staining with F-actin markers

• Correlate DOCK3 expression levels with fusion capacity in normal versus DMD myoblasts

Studies have shown that DMD myoblasts treated with shRNAi DOCK3 exhibit a significantly higher myogenic fusion index compared to controls, demonstrating DOCK3's importance in this process . Conversely, DOCK3 knockout myoblasts show impaired differentiation and reduced fusion capabilities .

How can researchers use DOCK3-FITC antibodies to study metabolic regulation in muscle?

DOCK3 knockout mice exhibit significant metabolic phenotypes, including hyperglycemia and altered glucose tolerance . To investigate DOCK3's metabolic functions:

• Use DOCK3-FITC antibodies to track protein localization in muscle biopsies from metabolic disease models

• Co-stain with SORBS1 antibodies to examine the DOCK3-SORBS1 interaction that regulates metabolism

• Correlate DOCK3 expression levels with measures of insulin sensitivity and glucose tolerance

• Analyze DOCK3 distribution in muscle fiber types with different metabolic profiles

• Compare DOCK3 staining patterns between healthy and diabetic muscle samples

• Investigate the relationship between DOCK3 and GLUT4 trafficking

This approach can reveal how DOCK3 contributes to skeletal muscle glucose homeostasis through its interaction with SORBS1 and potential effects on GLUT4 processing .

What technical challenges exist when using DOCK3-FITC antibodies for quantitative analysis?

Researchers should address several technical considerations for quantitative applications:

• Signal-to-noise ratio optimization: The FITC fluorophore has relatively high photobleaching rates compared to newer dyes

• Autofluorescence: Muscle tissue has significant autofluorescence that can interfere with FITC detection

• Fixing conditions: Different fixation methods can affect epitope accessibility and fluorescence intensity

• Antibody concentration: Titration is necessary as FITC conjugation can alter optimal working dilutions

• Subcellular localization: Distinguishing between membrane-bound and cytoplasmic DOCK3 requires high-resolution imaging

• Fluorescence quantification: Standardized protocols for intensity measurement are needed for comparative studies

For flow cytometry applications, use 5 μl of antibody per 10^6 cells in a 100 μl suspension as a starting point, similar to other FITC-conjugated antibodies .

How does DOCK3 expression change in dystrophic conditions?

DOCK3 exhibits altered expression patterns in muscular dystrophy:

• Increased DOCK3 expression correlates with disease severity in DMD patients

• DOCK3 acts as a dosage-sensitive biomarker of DMD progression

• Partial knockdown of DOCK3 (haploinsufficiency) improves muscle pathology in mdx mice

• Complete knockout of DOCK3 in dystrophin-deficient mice worsens skeletal muscle and cardiac phenotypes

These findings suggest an optimal DOCK3 expression level exists for muscle health, with both overexpression and complete absence being detrimental . DOCK3-FITC antibodies can be valuable tools for monitoring these expression changes in patient biopsies and experimental models.

How can DOCK3-FITC antibodies be used to evaluate therapeutic interventions?

DOCK3-targeting therapies show promise for muscular dystrophy treatment:

• Monitor changes in DOCK3 expression levels following therapeutic intervention

• Assess restoration of normal subcellular localization patterns

• Correlate DOCK3 levels with improvement in myofiber architecture and regeneration

• Evaluate normalization of DOCK3-interacting pathways (GSK-3β, RAC1, WAVE complex)

• Compare DOCK3 expression in responders versus non-responders to therapy

Studies in zebrafish models demonstrated that low-dose DOCK3 morpholino treatment improved muscle fiber architecture in dystrophic fish, while high-dose treatment worsened the phenotype, highlighting the importance of precise dosage monitoring .

What is the recommended protocol for immunofluorescence staining with DOCK3-FITC antibodies?

Optimized protocol for DOCK3 immunofluorescence:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature

• Washing: 3x5 minutes with PBS

• Permeabilization: 0.1% Triton X-100 for 5-10 minutes

• Blocking: 5% BSA in PBS for 1 hour at room temperature

• Primary antibody: DOCK3-FITC antibody at 5-15 μg/mL in blocking buffer, overnight at 4°C

• Washing: 3x5 minutes with PBS

• Counterstaining: DAPI (1:1000) for 5 minutes

• Mounting: Anti-fade mounting medium

• Imaging: Epifluorescence microscope with appropriate filter sets for FITC (Ex: 495nm, Em: 515-524nm)

For optimal results, protect samples from light during and after antibody incubation to prevent photobleaching of the FITC fluorophore.

What are common issues with DOCK3-FITC antibodies and how can they be resolved?

When troubleshooting, always include appropriate positive and negative controls to differentiate between antibody issues and biological variability .

How can researchers optimize multiplexing experiments with DOCK3-FITC antibodies?

For successful co-staining with other fluorophore-conjugated antibodies:

• Choose fluorophores with minimal spectral overlap (e.g., FITC + Cy5)

• If using multiple mouse or rabbit antibodies, consider sequential staining protocols

• Begin with DOCK3-FITC staining, followed by additional markers

• For optimizing DOCK3/GSK-3β co-staining, use antibodies against phosphorylated-GSK-3β from different host species

• When studying DOCK3/WAVE complex interactions, pair FITC (green) with red fluorophores

• Use spectral unmixing on confocal systems when fluorophore emission spectra overlap

• Validate multiplex protocols with single-stained controls to assess bleed-through

This approach allows simultaneous visualization of DOCK3 with its interaction partners or downstream targets in the same sample.