DOCK1 Antibody

What are the primary applications for DOCK1 antibodies in research?

DOCK1 antibodies are primarily utilized in Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), and ELISA applications. According to validation data from multiple sources, antibodies like catalog number ABIN1858660 have been specifically validated for WB, IHC, and ICC applications . For optimal results, researchers should consider the specific application requirements when selecting an antibody. For example, when performing Western blot analysis, dilutions typically range from 1:500-1:6000 depending on the specific antibody and sample type .

How should researchers select the appropriate DOCK1 antibody for their experiments?

Selection should be based on:

• Target specificity: Choose antibodies that target specific amino acid regions of DOCK1 relevant to your research. Available options include:

  • Antibodies targeting AA 1200-1435

  • Antibodies targeting AA 1676-1865

  • Antibodies targeting C-terminal regions

• Host and clonality: Consider whether rabbit polyclonal (more common for DOCK1) or mouse monoclonal antibodies are more suitable for your experimental design .

• Validated applications: Verify that the antibody has been validated for your specific application (WB, IHC, etc.) .

• Species reactivity: Confirm reactivity with your species of interest. Most DOCK1 antibodies react with human samples, but many also show cross-reactivity with mouse and rat samples .

What are the recommended protocols for Western blotting using DOCK1 antibodies?

For optimal Western blot results with DOCK1 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 6-8% gels due to DOCK1's high molecular weight (215 kDa, though it may run anomalously at 180-190 kDa)

• Transfer conditions:

  • Extended transfer times (90-120 min) at lower voltage or overnight transfer at 4°C are recommended for large proteins

• Antibody dilutions:

  • Primary antibody: 1:500-1:3000 (antibody-dependent)

  • Secondary antibody: Typically 1:5000-1:10000

• Detection method:

  • Enhanced chemiluminescence (ECL) is commonly used for visualizing bands

  • Expected band size: ~180-215 kDa

Note: When using 1 μg/mL of goat anti-human DOCK1 antibody, successful detection of the ~180 kDa band has been achieved in HUVEC, HepG2, C6, and PC-12 cell lines .

What is the optimal approach for immunohistochemical staining with DOCK1 antibodies?

For effective IHC with DOCK1 antibodies:

• Tissue preparation:

  • 4% paraformaldehyde fixation followed by paraffin embedding

  • 4-6 μm thick tissue sections

• Antigen retrieval:

  • Heat-mediated antigen retrieval using 10mM citrate buffer (pH 6.0)

  • 15-20 minutes at 95-100°C

• Blocking:

  • 5-10% normal serum (matching the species of secondary antibody)

  • 1 hour at room temperature

• Antibody dilutions:

  • Primary: 1:20-1:200 for IHC applications

  • Secondary: Follow manufacturer's recommendations

• Counterstaining:

  • Hematoxylin for nuclei visualization

  • Optional: DAPI for fluorescent applications

Validation studies have successfully used DOCK1 antibodies for IHC staining of human lung tissue at a dilution of 1:100 .

What is the biological function of DOCK1 in normal and pathological conditions?

DOCK1 (Dedicator of Cytokinesis 1) serves several critical cellular functions:

• Normal function:

  • Functions as a guanine nucleotide exchange factor (GEF) that activates Rac Rho small GTPases by exchanging bound GDP for free GTP

  • Regulates cytoskeletal rearrangements for cell motility and phagocytosis

  • Mediates cellular engulfment during apoptosis

  • Participates in embryonic development, axonogenesis, and angiogenesis

  • Forms complexes with ELMO1 to activate Rac GTPase for cytoskeletal reorganization

• Pathological implications:

  • Promotes cancer progression through multiple mechanisms:

    • Enhances malignant biological behavior of endometrial cancer cells via c-RAF/ERK1/2 signaling pathways

    • Regulates growth and motility through the RRP1B-DNMT-claudin-1 pathway in claudin-low breast cancer

    • Mediates EMT (epithelial-mesenchymal transition) in various cancers

    • Acts as a potential biomarker for cancer progression

How do knockout and overexpression of DOCK1 affect cellular functions?

Experimental modulation of DOCK1 expression reveals:

• DOCK1 knockout effects:

  • Inhibits proliferation, invasion, migration, and viability of cancer cells

  • Increases E-cadherin expression (promoting cell adhesion)

  • Decreases expression of MMP9, Ezrin, Bcl-2, p-c-RAF, and p-ERK1/2

  • Reduces tumor growth and weight in xenograft models

  • Impairs phagocytosis of apoptotic cells

• DOCK1 overexpression effects:

  • Enhances cancer cell proliferation, invasion, and migration

  • Downregulates E-cadherin expression

  • Upregulates MMP9, Ezrin, Bcl-2, p-c-RAF, and p-ERK1/2 expression

  • Accelerates tumor progression

These functional changes highlight DOCK1 as a potential therapeutic target in cancer treatment.

How can researchers address non-specific binding issues with DOCK1 antibodies?

Non-specific binding is a common challenge when using DOCK1 antibodies. Consider these strategies:

• Antibody validation:

  • Confirm specificity using positive and negative controls

  • Use peptide blocking experiments as demonstrated with HeLa cell lysates

  • Employ DOCK1 knockout or knockdown samples as negative controls

• Protocol optimization:

  • Increase blocking time/concentration (5-10% BSA or normal serum)

  • Optimize antibody dilutions (start with 1:1000 for WB)

  • Extend washing steps (4-5 washes of 10 minutes each)

  • Use detergents (0.1-0.3% Tween-20) in washing buffers

• Sample preparation:

  • Ensure complete protein denaturation for Western blotting

  • Use freshly prepared samples to minimize protein degradation

  • Include protease inhibitors in lysis buffers

• Signal detection:

  • Use highly specific secondary antibodies

  • Consider enzymatic/fluorescent secondary antibodies depending on background issues

What strategies can resolve inconsistent detection of DOCK1 in Western blotting?

Inconsistent detection of high molecular weight proteins like DOCK1 (~215 kDa) can be particularly challenging:

• Transfer optimization:

  • Use wet transfer systems for large proteins

  • Reduce transfer voltage and extend transfer time

  • Add SDS (0.1%) to transfer buffer to improve large protein elution

  • Consider using PVDF membranes instead of nitrocellulose for better protein retention

• Protein loading and separation:

  • Increase protein loading (40-60 μg)

  • Use lower percentage gels (6-8%) for better separation of high molecular weight proteins

  • Extend running time to improve separation

• Antibody considerations:

  • Test multiple DOCK1 antibodies targeting different epitopes

  • Fresh antibody aliquots may improve detection

  • Consider using enhanced detection systems (HRP polymers, amplification kits)

• Technical considerations:

  • DOCK1 may run anomalously at 180-190 kDa despite its calculated 215 kDa size

  • Confirm correct molecular weight using positive control lysates (e.g., HUVEC, HepG2, HeLa cells)

How can DOCK1 antibodies be utilized in studying cancer mechanisms?

Advanced applications of DOCK1 antibodies in cancer research include:

• Signaling pathway analysis:

  • Monitor DOCK1's role in the c-RAF/ERK1/2 signaling pathway in endometrial cancer

  • Investigate DOCK1-mediated regulation of RRP1B-DNMT-claudin-1 pathway in breast cancer

  • Analyze DOCK1's interaction with Rac1 and its downstream effectors

• Therapeutic target validation:

  • Combine DOCK1 antibodies with inhibitors like Raf inhibitor LY3009120 to assess pathway blockade

  • Use phospho-specific antibodies to monitor inhibition efficacy on downstream targets

• Biomarker development:

  • Employ IHC with DOCK1 antibodies for tumor specimen analysis

  • Correlate DOCK1 expression with cancer progression and patient outcomes

  • Develop tissue microarray analysis protocols for high-throughput screening

• Mechanistic studies:

  • Use co-immunoprecipitation with DOCK1 antibodies to identify novel binding partners

  • Implement chromatin immunoprecipitation (ChIP) to study DOCK1's role in transcriptional regulation

  • Employ proximity ligation assays to visualize DOCK1 interactions with signaling molecules

What are the methodological considerations for co-immunoprecipitation experiments using DOCK1 antibodies?

For successful co-immunoprecipitation (Co-IP) studies with DOCK1:

• Sample preparation:

  • Use gentle lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain post-translational modifications

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Antibody selection:

  • Choose high-affinity DOCK1 antibodies with minimal cross-reactivity

  • Consider using monoclonal antibodies for higher specificity

  • Test multiple antibodies targeting different DOCK1 epitopes

• Experimental controls:

  • Include IgG control from the same species as the DOCK1 antibody

  • Use DOCK1-depleted cells as negative controls

  • Include known DOCK1 interactors (e.g., ELMO1) as positive controls

• Detection strategies:

  • Perform reciprocal Co-IPs when possible

  • Consider crosslinking antibodies to beads to prevent IgG contamination

  • Use clean detection antibodies that don't cross-react with the IP antibody

• Data analysis:

  • Quantify the ratio of co-precipitated protein to immunoprecipitated DOCK1

  • Normalize to input controls

  • Consider using mass spectrometry for unbiased identification of novel interactions

How can DOCK1 antibodies be used in combination with modern imaging techniques?

Advanced imaging approaches with DOCK1 antibodies include:

• Super-resolution microscopy:

  • STORM/PALM microscopy to visualize DOCK1 localization at nanometer resolution

  • Determine co-localization with binding partners at membrane protrusions

  • Image DOCK1 recruitment during cell migration or phagocytosis

• Live-cell imaging:

  • Use anti-DOCK1 Fab fragments conjugated to quantum dots

  • Track DOCK1 dynamics during cellular processes

  • Implement FRET-based assays to study DOCK1-partner interactions

• Tissue imaging:

  • Multiplex immunofluorescence to analyze DOCK1 co-expression with other markers

  • Spatial transcriptomics combined with DOCK1 protein localization

  • Digital pathology analysis of DOCK1 expression patterns in tissue microarrays

• Subcellular localization:

  • Immuno-electron microscopy for ultrastructural localization

  • Correlative light and electron microscopy (CLEM) to link DOCK1 function to ultrastructure

  • Expansion microscopy for enhanced visualization of protein complexes

What are the considerations for designing antibody-based therapeutic approaches targeting DOCK1?

The translational potential of anti-DOCK1 strategies includes:

• Therapeutic antibody development:

  • Generate antibodies that inhibit DOCK1-mediated GEF activity

  • Target critical domains like DHR-2 (aa 1111-1616) responsible for GTPase binding

  • Develop internalizing antibodies for intracellular delivery of therapeutics

• Delivery challenges:

  • Consider antibody formats that can access intracellular DOCK1 (e.g., cell-penetrating antibodies)

  • Explore antibody-drug conjugates targeting DOCK1-expressing cells

  • Implement nanoparticle-based delivery systems for enhanced cellular uptake

• Efficacy assessment:

  • Monitor downstream signaling (Rac activation, ERK phosphorylation)

  • Assess phenotypic changes in cell motility and invasion

  • Evaluate effects on tumor growth in preclinical models

• Translation considerations:

  • Evaluate therapeutic window based on differential expression between normal and cancer cells

  • Assess potential immune-related adverse events

  • Design combination strategies with established cancer therapies

How should researchers interpret variations in DOCK1 expression levels across different tissue types?

Interpreting DOCK1 expression patterns requires systematic analysis:

• Baseline expression:

  • DOCK1 is generally expressed in non-hematopoietic cell types

  • Expression levels vary across normal tissues and their development stages

  • Consider using tissue-specific positive controls for accurate comparison

• Methodological considerations:

  • Standardize quantification methods (e.g., densitometry for Western blots)

  • Use housekeeping proteins appropriate for the tissue being studied

  • Implement quantitative approaches like fluorescence intensity measurements for IHC/IF

• Pathological contexts:

  • DOCK1 expression is increased in endometrial cancer tissues compared to adjacent normal tissues

  • Similar upregulation patterns observed in other cancer types

  • Correlate expression with clinical parameters and outcomes

• Experimental validation:

  • Confirm antibody-based results with orthogonal methods (e.g., qPCR, mass spectrometry)

  • Consider cell type heterogeneity within tissue samples

  • Account for post-translational modifications that may affect antibody binding

What strategies can help researchers integrate DOCK1 antibody data with other -omics approaches?

Multi-omics integration of DOCK1 antibody data:

• Transcriptomics integration:

  • Compare protein expression (antibody-based) with mRNA levels

  • Analyze discrepancies that might indicate post-transcriptional regulation

  • Utilize RNA-seq data to identify DOCK1 isoforms that may affect antibody binding

• Proteomics combination:

  • Complement targeted antibody approaches with unbiased mass spectrometry

  • Identify post-translational modifications using modification-specific antibodies

  • Map the DOCK1 interactome through affinity purification-mass spectrometry

• Functional genomics correlation:

  • Integrate CRISPR/Cas9 screening data with DOCK1 expression analysis

  • Correlate genetic alterations in DOCK1 with protein expression patterns

  • Use antibody-based readouts to validate genomic findings

• Computational analysis:

  • Implement machine learning approaches to identify patterns in multi-omics data

  • Develop predictive models incorporating DOCK1 expression with other molecular features

  • Utilize pathway enrichment analysis to contextualize DOCK1 function

How should researchers design experiments to study DOCK1's role in specific signaling pathways?

Rigorous experimental design for DOCK1 signaling studies:

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated DOCK1 knockout as demonstrated in endometrial cancer cells

  • siRNA/shRNA knockdown for transient depletion

  • Overexpression systems using vectors containing human DOCK1 cDNA

  • Domain-specific mutants to dissect functional regions

• Pharmacological interventions:

  • Combine with pathway-specific inhibitors like Raf inhibitor LY3009120

  • Assess Rac1 inhibitors to establish downstream dependency

  • Use time-course analysis to determine signaling dynamics

• Readout methods:

  • Phosphorylation status of pathway components (c-RAF, ERK1/2)

  • Expression of downstream targets (E-cadherin, MMP9, Ezrin, Bcl-2)

  • Functional assays: proliferation (BrdU), invasion (transwell), apoptosis (flow cytometry)

• Validation approaches:

  • Rescue experiments to confirm specificity

  • In vivo models (xenografts) to validate in vitro findings

  • Alternative pathway activation to assess specificity

What are the optimal approaches for studying DOCK1 in in vivo models?

In vivo experimental design for DOCK1 research:

• Model selection:

  • Xenograft models using DOCK1-modulated cell lines

  • Genetically engineered mouse models with tissue-specific DOCK1 alterations

  • Patient-derived xenografts for translational relevance

• Intervention approaches:

  • Orthotopic implantation for tissue-specific microenvironment

  • Timing considerations for intervention (prevention vs. treatment)

  • Combination with standard-of-care treatments

• Analysis methods:

  • Tumor volume and weight measurements

  • IHC analysis of DOCK1 and downstream targets in tumor sections

  • Metastasis assessment through ex vivo imaging

• Technical considerations:

  • Ensure antibody specificity in mouse tissues

  • Use appropriate controls (scrambled shRNA, empty vector)

  • Account for tumor heterogeneity in analysis

The endometrial cancer xenograft models have successfully demonstrated that DOCK1 knockout inhibits tumor growth and alters expression of E-cadherin, MMP9, Ezrin, and Bcl-2 in vivo .