DOK5 Antibody

What is DOK5 and what are its primary cellular functions?

DOK5 (Docking Protein 5) belongs to the DOK family of membrane proteins that function as adapter proteins in signal transduction pathways. The protein contains tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at its amino terminus that facilitate protein-protein interactions .

DOK5 serves multiple functions:

• Acts as a substrate for insulin and insulin-like growth factor signaling (also known as IRS6)

• Functions in RET-mediated neurite outgrowth

• Plays a positive role in activation of the MAP kinase pathway

• Regulates cell proliferation and differentiation pathways

• Participates in intracellular signaling pathways that influence cellular growth

Unlike other DOK family proteins, DOK5 does not interact with RasGAP, suggesting a distinct signaling mechanism .

How does DOK5 expression vary across different tissues and cell types?

Based on research findings, DOK5 shows differential expression patterns across tissues:

• Neural tissues: Expressed in brain tissue and involved in neuronal differentiation via RET signaling

• Immune cells: Present in scattered cells in the interfollicular area of tonsil, compatible with mast cells

• Gastric tissue: Variably expressed in gastric cancer tissues

• Skeletal system: Expressed in osteoblasts where it regulates differentiation

• Fetal tissues: Detected in fetal brain and fetal heart lysates via Western blot

Research has shown the capability of detecting DOK5 in mouse kidney, mouse liver, and rat skeletal muscle as positive samples for antibody validation .

What criteria should be considered when selecting a DOK5 antibody for research applications?

When selecting a DOK5 antibody, researchers should consider:

• Target epitope specificity: Different antibodies target different regions (N-terminal, C-terminal, or specific internal domains). For example:

  • N-terminal targeting antibodies (ABIN359962)

  • C-terminal targeting antibodies (AP7693B, aa 291-306)

  • Internal domain targeting (CAB13726, aa 237-306)

• Host species and clonality:

  • Rabbit polyclonal antibodies offer broad epitope recognition

  • Rabbit monoclonal antibodies (EPR9923(B)) provide higher specificity and consistency

  • Goat polyclonal antibodies offer alternative validation approaches

• Validated applications:

  • Western blotting (typically 1:500-1:2000 dilution)

  • IHC-P (typically 1:50-1:100 dilution)

  • ELISA (typically 1:20000 dilution)

• Species reactivity: Confirm cross-reactivity with your experimental model:

  • Human-only reactivity

  • Human/mouse reactivity

  • Human/mouse/rat reactivity

• Validation data: Choose antibodies with extensive validation data relevant to your application .

How can I validate a DOK5 antibody for my specific experimental system?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Use tissues known to express DOK5 (mouse kidney, mouse liver, rat skeletal muscle)

  • Use transfected vs. non-transfected cell lines: In HEK293 cells transiently expressing DOK5, a band of approximately 38kDa is observed that is absent in non-transfected cells

• Knockdown validation:

  • Use DOK5 shRNA to create knockdown models (three validated shRNA sequences are available) :

• Molecular weight verification:

  • The predicted molecular weight of DOK5 is 35 kDa based on sequence analysis

  • Observed molecular weight in Western blot ranges from 30-38 kDa depending on the system

• Cross-platform validation:

  • Validate with orthogonal approaches (e.g., combining Western blot with IHC or IF)

  • Sequence-based verification using multiple antibodies targeting different epitopes

• Reproducibility testing:

  • Test antibody performance across different lots

  • Validate antibody in multiple model systems when applicable

What are the optimal conditions for Western blotting with DOK5 antibodies?

For optimal Western blotting with DOK5 antibodies:

• Sample preparation:

  • Use RIPA buffer supplemented with 1% protease inhibitor and PMSF on ice for 30 minutes

  • Centrifuge at 12,000 rpm for 10 minutes at 4°C to eliminate cell debris

  • Load 10-25 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 10% SDS-PAGE for optimal separation

  • Transfer to PVDF membranes at standard conditions

• Blocking and antibody incubation:

  • Block with 5% skimmed milk at room temperature for 2 hours

  • Primary antibody dilutions:

    • Rabbit polyclonal: 1:500-1:2000

    • Rabbit monoclonal: 1:10000

  • Incubate with primary antibody overnight at 4°C with shaking

  • Wash thrice with TBST, 15 minutes each time

  • Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit HRP at 1:2000) at room temperature for 1 hour

• Detection:

  • Develop using ECL chemiluminescence system

  • Expected band size: approximately 35 kDa (30-38 kDa observed in different systems)

• Positive controls:

  • IM9 cell lysate

  • Human fetal brain lysate

  • Human fetal heart lysate

  • Mouse liver tissue

How can DOK5 antibodies be used effectively in immunohistochemistry?

For effective immunohistochemistry with DOK5 antibodies:

• Sample preparation:

  • Paraffin-embedded sections are recommended

  • Standard deparaffinization and rehydration protocols apply

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective

  • Some antibodies may require EDTA-based retrieval (pH 9.0) - check manufacturer's recommendations

• Blocking and antibody incubation:

  • Block with appropriate serum (typically 5-10% normal serum from secondary antibody host species)

  • Primary antibody dilutions:

    • For rabbit polyclonal: 1:50-1:100

    • For goat polyclonal: 1-2 μg/ml recommended concentration

  • Incubate overnight at 4°C or 1-2 hours at room temperature

• Visualization:

  • Use appropriate detection system (HRP/DAB or fluorescent secondary)

  • Include DAPI counterstain for fluorescent detection

• Expected results:

  • In tonsil tissue, DOK5 stains scattered cells in the interfollicular area compatible with mast cells

  • In gastric cancer tissues, DOK5 expression correlates with immune cell infiltration patterns

• Controls:

  • Include isotype controls to rule out non-specific binding

  • Include known positive tissue controls based on experimental goals

What is the role of DOK5 in cancer, particularly gastric cancer?

DOK5 has emerged as a significant player in cancer biology, particularly in gastric cancer (GC):

Interestingly, DOK5 shows tissue-specific prognostic patterns; while high expression correlates with poor prognosis in gastric cancer, low expression correlates with poor prognosis in liver cancer .

How is DOK5 involved in neurological disorders and neural development?

DOK5 has been implicated in several neurological contexts:

• Bipolar disorder:

  • A genome-wide association study found that an SNP in DOK5 (rs2023454) was strongly associated with right amygdala activation under hostility contrast (p = 4.88 × 10^-7, false discovery rate = 0.05)

  • This SNP accounted for approximately 33% of the variance in youths with bipolar disorder and 12% of the variance in healthy youths

  • DOK5 encodes a substrate of tropomyosin-related kinase B/C receptors involved in neurotrophin signaling

• Neural development:

  • DOK5 functions in RET-mediated neurite outgrowth

  • Acts as a putative link with downstream effectors of RET in neuronal differentiation

  • As an adapter protein, DOK5 helps assemble multimolecular signaling complexes crucial for neural development

• Signaling pathways:

  • Plays a positive role in activation of the MAP kinase pathway, which is critically important in neural development and function

  • Unlike other DOK family proteins, DOK5 does not interact with RasGAP, suggesting a specialized function in neural contexts

These findings indicate DOK5 may be a potential target for understanding and potentially treating certain neurological disorders, particularly those involving amygdala function.

How can DOK5 antibodies be used in single-cell analysis techniques?

While the search results don't specifically address single-cell applications for DOK5 antibodies, researchers can adapt established protocols:

• Single-cell Western blotting:

  • Use validated DOK5 antibodies (1:500-1:2000 dilution) with microfluidic platforms

  • Verify specificity with positive controls (e.g., IM9 cells, neural tissues)

  • Combine with other neural or cancer markers for multiparametric analysis

• Mass cytometry (CyTOF):

  • Metal-conjugated DOK5 antibodies can be incorporated into CyTOF panels

  • Focus on validated clones with high specificity (e.g., EPR9923(B))

  • Design panels to simultaneously assess DOK5 with immune markers shown to correlate with its expression in cancer contexts

• Single-cell RNA-seq validation:

  • Use DOK5 antibodies in immunofluorescence to validate scRNA-seq findings

  • Apply for spatial transcriptomics validation where DOK5 expression patterns are of interest

  • Develop antibody-based sorting strategies to isolate DOK5-positive cell populations for downstream genomic analysis

• Live-cell imaging:

  • While current data focuses on fixed-cell applications, researchers could explore membrane-permeable DOK5 antibody derivatives for live-cell tracking

  • Consider developing fluorescent protein-tagged DOK5 constructs as alternatives for dynamic studies

What is the relationship between DOK5 and cellular differentiation in non-cancer contexts?

Beyond cancer, DOK5 plays important roles in cellular differentiation:

• Osteoblast differentiation:

  • DOK5 knockdown suppresses osteoblastic differentiation and the expression of osteogenic biosignatures

  • Three validated shRNA sequences have been established for DOK5 knockdown studies in osteoblast models:

• Fibrosis and tissue remodeling:

  • DOK5 is upregulated in systemic sclerosis and associated with IGFBP-5-induced fibrosis

  • This suggests a role in aberrant tissue remodeling and fibrotic disease processes

• Insulin signaling in metabolic tissues:

  • As insulin receptor substrate 6 (IRS6), DOK5 participates in metabolic signaling

  • May influence differentiation and function of metabolic tissues through insulin and insulin-like growth factor signaling

• Neural differentiation:

  • Functions in RET-mediated neurite outgrowth

  • Serves as a putative link with downstream effectors of RET in neuronal differentiation

  • May influence neural progenitor cell fate decisions through these pathways

Understanding these non-cancer roles of DOK5 may provide insights into developmental biology and potential regenerative medicine applications.

What advanced bioinformatic approaches can be used to study DOK5 in the context of immune cell infiltration?

Based on the gastric cancer research findings, several advanced bioinformatic approaches can be applied to study DOK5's relationship with immune infiltration:

• Integrated multi-omics analysis:

  • Combine transcriptomics (DOK5 expression), proteomics (DOK5 protein levels), and immunophenotyping data

  • Use tools like TIMER database to examine correlations between DOK5 expression and immune cell infiltration across 32 cancer types

  • Analyze DOK5 somatic copy number alterations (SCNA) in relation to infiltration levels of CD8+ T cells, B cells, neutrophils, macrophages, and dendritic cells

• Correlation network analysis:

  • Build networks of DOK5 and immune markers using GEPIA and TIMER databases

  • Focus on established correlations with markers for:

    • T cells (general, Th1, Th2, Tfh, Th17, Treg, exhausted)

    • B cells

    • Macrophages (M1, M2, TAM)

    • Monocytes

    • Neutrophils

    • NK cells

    • Dendritic cells

• Pathway enrichment analysis:

  • Apply GSEA to identify signaling pathways associated with DOK5 expression

  • Perform GO and KEGG analyses to understand biological processes enriched in genes positively associated with DOK5

  • Key findings have shown enrichment in:

    • Immune response and inflammatory response (biological processes)

    • Cytokine-cytokine receptor interaction

    • TNF signaling pathway

    • Transcriptional misregulation in cancer

• Survival analysis stratification:

  • Use Kaplan-Meier plotter and GEPIA to assess the prognostic impact of DOK5 in different contexts

  • Stratify analyses by:

    • Cancer type (gastric vs. liver cancer shows opposite patterns)

    • Immune infiltration levels

    • Molecular subtypes

These approaches can generate testable hypotheses about DOK5's role in immune modulation that can be validated experimentally using DOK5 antibodies.

How can I address non-specific binding or background issues when using DOK5 antibodies?

When encountering high background or non-specific binding with DOK5 antibodies:

• Antibody dilution optimization:

  • Titrate antibody concentrations more carefully (e.g., test a dilution series from 1:200 to 1:5000 for Western blot)

  • Different antibodies have recommended ranges:

    • For Western blot: 1:100-1:500 , 1:500-1:2000 , or 1:10000 depending on the antibody

    • For IHC: 1:50-1:100 or 1-2 μg/ml

• Blocking optimization:

  • Increase blocking duration (from 1 hour to 2 hours or overnight)

  • Try alternative blocking agents (5% BSA instead of milk for phospho-specific applications)

  • For tissues with high endogenous biotin, use avidin-biotin blocking system if using biotin-based detection

• Washing protocol adjustment:

  • Increase washing duration (e.g., 15 minutes per wash instead of 5 minutes)

  • Add 0.1-0.3% Triton X-100 to wash buffers for improved membrane penetration

  • Increase the number of washes (5-6 washes instead of 3)

• Alternative antibody selection:

  • Consider antibodies targeting different epitopes:

    • N-terminal targeting (ABIN359962)

    • C-terminal targeting (AP7693B, aa 291-306)

    • Internal domain targeting (CAB13726, aa 237-306)

  • Switch from polyclonal to monoclonal antibodies (EPR9923(B)) for increased specificity

• Validation with biological controls:

  • Include DOK5 knockdown samples using validated shRNAs

  • Use non-expressing cell lines as negative controls (e.g., non-transfected HEK293)

What are the common pitfalls in interpreting DOK5 expression data and how can they be avoided?

Researchers should be aware of several challenges when interpreting DOK5 expression data:

• Tissue-specific expression patterns:

  • DOK5 shows variable expression across tissues and may have tissue-specific functions

  • Solution: Always include tissue-relevant positive controls and interpret findings within the specific tissue context

• Contradictory prognostic associations:

  • High DOK5 expression correlates with poor prognosis in gastric cancer but better prognosis in liver cancer

  • Solution: Avoid generalizing findings from one cancer type to another; always validate in the specific disease context

• Multiple protein isoforms:

  • DOK5 may have tissue-specific isoforms or post-translational modifications

  • Solution: Use antibodies targeting different epitopes and verify band sizes carefully (expected range: 30-38 kDa)

• Immune infiltration confounding:

  • DOK5 expression correlates strongly with immune cell infiltration, which may confound interpretation in heterogeneous tissue samples

  • Solution: Use single-cell approaches or microdissection to distinguish tumor-specific from immune-cell-specific expression

• Cross-reactivity concerns:

  • DOK family members share sequence homology which may lead to cross-reactivity

  • Solution: Validate antibody specificity against other DOK family members (DOK1-7) using overexpression systems or CRISPR knockout models

• Variable subcellular localization:

  • DOK5 can be found in both cytoplasmic and membrane locations depending on activation state

  • Solution: Use subcellular fractionation or high-resolution imaging to determine precise localization in your experimental system