DOT3 Antibody

What is DOK3 and what cellular functions does it regulate?

DOK3 (Docking protein 3, also known as Downstream of tyrosine kinase 3) belongs to the DOK family of adaptor proteins. These proteins are enzymatically inert but serve as critical scaffolding molecules that provide docking platforms for the assembly of multimolecular signaling complexes. DOK3 functions primarily as a negative regulator of JNK signaling in B-cells through its interaction with INPP5D/SHIP1. Additionally, research suggests that DOK3 may modulate ABL1 function in cellular signaling pathways .

The DOK family proteins play essential roles in immune cell signaling, with DOK3 specifically contributing to:

• Negative regulation of B-cell receptor signaling

• Modulation of tyrosine kinase-dependent pathways

• Coordination of protein-protein interactions in immune response

• Potential roles in cellular proliferation and differentiation processes

What applications are DOK3 antibodies validated for in research settings?

DOK3 antibodies, such as the rabbit polyclonal ab236609, have been validated for multiple research applications in both human and mouse experimental systems. These include:

• Western blotting (WB): Successfully used at 1/500 dilution with mouse heart and placenta tissue lysates, producing bands at the expected molecular weight of 53 kDa

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Effectively detects DOK3 in human tonsil tissue at 1/100 dilution

• Immunocytochemistry/Immunofluorescence (ICC/IF): Validated for cellular localization studies in various cell types including human lung carcinoma cell lines

Each application requires specific optimization of antibody concentration, incubation conditions, and detection systems depending on the experimental context and tissue/cell source.

How should I design proper controls for DOK3 antibody experiments?

When designing experiments with DOK3 antibodies, several controls are essential for result validation:

• Positive controls: Include tissues or cell lines known to express DOK3, such as:

  • Mouse heart tissue for Western blot applications

  • Human tonsil tissue for IHC-P applications

  • Human lymphoid cell lines for functional studies

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at matching concentration)

  • Tissues/cells with confirmed low/no DOK3 expression

• Specificity controls:

  • Peptide competition assay using the immunizing peptide (DOK3 aa 1-150)

  • Knockdown/knockout validation in cell lines using siRNA or CRISPR methods

  • Recombinant protein expression systems for antibody validation

All experiments should include appropriate loading controls (β-actin, GAPDH) for Western blots and tissue-specific markers for IHC applications to ensure consistent sample preparation and processing.

How can I optimize antibody concentration for DOK3 detection in different tissue types?

Optimization of DOK3 antibody concentration is tissue-dependent and requires systematic titration. Based on existing research protocols:

• For Western blot applications:

  • Begin with 1/500 dilution as a starting point based on validated applications

  • Prepare a dilution series (1/250, 1/500, 1/1000, 1/2000) to determine optimal signal-to-noise ratio

  • Different tissues may require different concentrations; for instance, lymphoid tissues might need lower concentrations due to higher DOK3 expression

  • Optimize blocking conditions with 5% non-fat milk or BSA to minimize background

• For IHC-P applications:

  • Initial dilution of 1/100 is recommended

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Optimize incubation time and temperature (overnight at 4°C vs. 1-2 hours at room temperature)

  • Consider signal amplification systems for tissues with low expression levels

• For ICC/IF applications:

  • Begin with 1/100-1/200 dilution

  • Test different fixation methods (4% paraformaldehyde, methanol, acetone)

  • Adjust permeabilization conditions depending on subcellular localization

The antibody biodistribution coefficient (ABC) concept suggests that the relationship between plasma and tissue concentrations of antibodies may follow predictable patterns across species , which could inform tissue-specific optimization strategies.

What approaches can resolve contradictory results when using DOK3 antibodies in different experimental systems?

When facing contradictory results with DOK3 antibodies across different experimental systems, consider these methodological approaches:

• Epitope accessibility issues:

  • The DOK3 antibody (ab236609) targets amino acids 1-150 ; certain experimental conditions may affect epitope exposure

  • Test multiple antibodies targeting different DOK3 epitopes

  • Vary denaturation conditions in Western blots (reducing vs. non-reducing)

  • Explore alternative antigen retrieval methods for IHC/ICC

• Expression level variations:

  • Quantify baseline DOK3 expression in your experimental systems using qPCR

  • Consider cell activation state, as DOK proteins can be dynamically regulated during immune cell activation

  • Analyze post-translational modifications that might affect antibody recognition

• Cross-reactivity assessment:

  • Perform parallel experiments with DOK1 and DOK2 antibodies to rule out cross-reactivity

  • Include siRNA knockdown controls specific to each DOK family member

  • Employ recombinant DOK3 protein as competitive inhibitor

• Species-specific considerations:

  • Although DOK3 antibody reacts with both human and mouse samples , subtle species-specific differences in recognition may exist

  • Consider sequence alignment analysis between species when interpreting results

  • Test species-specific positive controls in parallel with experimental samples

How do post-translational modifications affect DOK3 antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) significantly impact DOK3 antibody recognition, potentially affecting experimental interpretation:

• Phosphorylation effects:

  • DOK3 contains multiple tyrosine phosphorylation sites that are dynamically regulated during cell signaling

  • Phosphorylation may mask or expose antibody epitopes, particularly for antibodies targeting regions containing phosphorylation sites

  • Consider using phosphorylation-specific DOK3 antibodies to distinguish activation states

  • Compare results with lambda phosphatase-treated samples to evaluate phosphorylation dependence

• Ubiquitination and proteasomal degradation:

  • DOK proteins undergo ubiquitin-dependent degradation that affects their stability and detection

  • Proteasome inhibitors (MG132, lactacystin) can be used to determine if protein degradation affects detection

  • Compare fresh samples with those subject to various storage conditions to assess degradation impact

• Experimental considerations for PTM analysis:

  • Include phosphatase inhibitors in lysis buffers when studying phosphorylated DOK3

  • Consider sample preparation timing, as PTMs can change rapidly during cell processing

  • Use MS/MS analysis to identify specific modifications present on DOK3 in your experimental system

  • Implement proximity ligation assays to study interactions dependent on specific modifications

How can DOK3 antibodies be utilized to study protein-protein interactions in immune signaling pathways?

DOK3 antibodies can be powerful tools for studying protein-protein interactions in immune signaling through these methodological approaches:

• Co-immunoprecipitation (Co-IP) protocols:

  • Use DOK3 antibody for immunoprecipitation at 1:50 dilution in cell lysates

  • Analyze precipitates for known interacting partners such as INPP5D/SHIP1

  • Cross-linking prior to lysis can capture transient interactions

  • Validate interactions with reciprocal Co-IP using antibodies against predicted binding partners

• Proximity-based interaction methods:

  • Proximity ligation assay (PLA) to visualize DOK3 interactions within intact cells

  • FRET/BRET approaches with fluorescently tagged DOK3 to study dynamic interactions

  • BioID or APEX2 proximity labeling with DOK3 fusion proteins to identify novel interacting partners

• Functional interaction mapping:

  • Create domain deletion constructs of DOK3 to map specific interaction regions

  • Use phosphorylation-deficient mutants to determine phosphorylation-dependent interactions

  • Apply CRISPR-Cas9 editing to modify endogenous DOK3 interaction domains

  • Combine with B-cell activation assays to correlate interactions with functional outcomes

Research on DR3-TL1A signaling pathways demonstrates how receptor-ligand interactions can be effectively studied using similar antibody-based approaches , providing methodological guidance for DOK3 interaction studies.

What are the considerations for using DOK3 antibodies in multiplex immunofluorescence applications?

Multiplex immunofluorescence with DOK3 antibodies requires careful planning to achieve specific labeling and avoid cross-reactivity:

• Antibody selection and validation:

  • Ensure primary antibodies are raised in different host species to avoid cross-reactivity

  • Validate each antibody individually before combining in multiplex panels

  • Test sequential versus simultaneous staining protocols to identify optimal conditions

  • Include single-color controls for spectral unmixing and compensation

• Panel design considerations:

  • For studying B-cell signaling, combine DOK3 with B-cell markers (CD19, CD20) and signaling proteins (SHIP1, SYK)

  • Use subcellular markers to evaluate DOK3 localization during cell activation

  • Consider using directly conjugated primary antibodies to minimize cross-reactivity

  • Include nuclear counterstains compatible with your fluorophore selection

• Technical optimization strategies:

  • Block with species-specific secondary antibody host serum to reduce background

  • Employ tyramide signal amplification for detection of low-abundance proteins

  • Use spectral imaging and unmixing to resolve overlapping fluorescence spectra

  • Consider tissue autofluorescence quenching methods for tissue sections

• Data analysis approaches:

  • Implement quantitative colocalization analysis to assess protein interactions

  • Use high-content imaging systems for large-scale, multiparametric data collection

  • Apply machine learning algorithms for pattern recognition in complex datasets

  • Consider spatial statistics to analyze distribution patterns of DOK3 relative to other proteins

How can researchers effectively differentiate between DOK family members in experimental systems?

Differentiating between the highly homologous DOK family members requires specific experimental strategies:

• Antibody specificity assessment:

  • Perform Western blots with recombinant DOK1, DOK2, and DOK3 proteins to confirm specificity

  • Use knockout/knockdown cell lines for each DOK family member as validation controls

  • Consider epitope mapping to identify antibodies targeting unique regions of DOK3

  • Test cross-reactivity with other DOK family members in overexpression systems

• Expression analysis methods:

  • Use qPCR with isoform-specific primers to quantify DOK1, DOK2, and DOK3 mRNA levels

  • Perform single-cell RNA-seq to characterize expression patterns across cell types

  • Design Northern blot probes targeting unique regions of DOK3 transcripts

  • Consider reporter cell lines with DOK3 promoter-driven fluorescent proteins

• Functional discrimination approaches:

  • Design siRNAs targeting unique regions of DOK3 mRNA

  • Use CRISPR-Cas9 genome editing with guide RNAs specific to DOK3

  • Perform rescue experiments with DOK3 constructs resistant to siRNA targeting

  • Develop isoform-specific functional assays based on known differential activities

• Structural biology approaches:

  • Use computational structural analysis to identify unique surface features of DOK3

  • Design peptide competitors based on unique DOK3 sequences

  • Consider developing nanobodies or aptamers with enhanced specificity for DOK3

  • Apply hydrogen-deuterium exchange mass spectrometry to map antibody binding sites

How should researchers address inconsistent DOK3 antibody staining patterns in immunohistochemistry?

Inconsistent DOK3 staining patterns in IHC applications can be addressed through systematic troubleshooting:

• Tissue processing and fixation variables:

  • Standardize fixation protocol (duration, fixative composition, temperature)

  • Compare freshly fixed tissues with archived specimens to assess degradation effects

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Evaluate the impact of section thickness on staining patterns

• Antibody validation approaches:

  • Test multiple DOK3 antibodies targeting different epitopes

  • Use blocking peptides to confirm specificity of observed staining

  • Include known positive and negative control tissues in each experiment

  • Consider dual staining with another method (e.g., RNA in situ hybridization) to confirm expression patterns

• Technical optimization strategies:

  Parameter	Variables to Test	Evaluation Method
  Antibody dilution	1:50, 1:100, 1:200, 1:500	Signal-to-noise ratio
  Antigen retrieval	Citrate pH 6.0, EDTA pH 9.0, Tris-EDTA pH 8.0	Staining intensity and specificity
  Incubation time	1h RT, overnight 4°C	Staining consistency
  Detection system	HRP-polymer, ABC, TSA amplification	Sensitivity and background

• Biological interpretation considerations:

  • DOK3 expression may vary with tissue activation state or disease progression

  • Consider cell-type specific expression patterns that might appear as "inconsistent" staining

  • Evaluate correlation with known DOK3-interacting proteins to confirm biological relevance

  • Document subcellular localization patterns to distinguish specific from non-specific staining

What strategies can resolve high background issues in Western blots using DOK3 antibodies?

High background in Western blots with DOK3 antibodies can be resolved through these methodological approaches:

• Sample preparation optimization:

  • Use fresh protease inhibitors in lysis buffers

  • Centrifuge lysates at high speed to remove insoluble debris

  • Determine optimal protein loading amount (typically 20-40 μg)

  • Consider using specialized lysis buffers for membrane-associated proteins

• Blocking and antibody incubation parameters:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Increase blocking time (1-3 hours at room temperature)

  • Dilute primary antibody in fresh blocking solution

  • Extend washing steps (5 x 5 minutes with 0.1% TBST)

• Systematic troubleshooting approach:

  Issue	Potential Cause	Solution
  Non-specific bands	Cross-reactivity	Use more stringent washing conditions
  General background	Insufficient blocking	Increase blocking time/concentration
  Membrane artifacts	Improper handling	Use clean forceps, avoid membrane drying
  Edge effects	Uneven antibody exposure	Ensure complete membrane submersion

• Advanced techniques for difficult samples:

  • Consider using gradient gels for better protein separation

  • Test nitrocellulose versus PVDF membranes for optimal binding

  • Pre-adsorb antibody with cell/tissue lysate from negative control samples

  • Implement signal enhancing systems for detection of low-abundance proteins

How can researchers accurately interpret DOK3 expression data in the context of immune cell activation states?

Accurate interpretation of DOK3 expression in immune cell activation requires consideration of multiple factors:

• Temporal dynamics and activation context:

  • Perform time-course experiments to capture dynamic changes in DOK3 levels

  • Compare multiple activation stimuli (antigen receptor engagement, cytokines, TLR ligands)

  • Correlate DOK3 expression/phosphorylation with known activation markers

  • Consider cell subset-specific responses using flow cytometry or single-cell approaches

• Post-translational regulation assessment:

  • Distinguish between total DOK3 protein levels and phosphorylated forms

  • Analyze ubiquitination state to assess protein stability during activation

  • Investigate subcellular relocalization that may affect functional activity

  • Consider protein-protein interactions that may sequester or expose DOK3

• Integrated data analysis approach:

  • Correlate DOK3 protein levels with mRNA expression

  • Perform parallel analysis of DOK3-interacting proteins

  • Assess functional outcomes of DOK3 modulation during activation

  • Use systems biology approaches to place DOK3 in signaling networks

• Experimental design considerations:

  • Include appropriate time-matched controls for each activation condition

  • Consider the impact of cell culture conditions on basal activation state

  • Use primary cells when possible, as cell lines may have altered signaling pathways

  • Implement genetic approaches (CRISPR, shRNA) to confirm DOK3-dependent effects

Research on regulatory T cell activation through DR3-TL1A axis demonstrates how activation marker analysis (ICOS, KLRG-1, PD-1, CD103, Ki-67) can be effectively integrated to understand immune cell activation states , providing a model for DOK3 activation studies.

How might DOK3 antibodies be utilized in single-cell analysis techniques?

DOK3 antibodies can be adapted for cutting-edge single-cell applications through these methodological approaches:

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated DOK3 antibodies enable high-dimensional analysis with 40+ parameters

  • Panel design should include lineage markers, activation markers, and other signaling proteins

  • Phospho-specific DOK3 antibodies can track signaling at single-cell resolution

  • Data analysis using viSNE, SPADE, or FlowSOM algorithms to identify cell populations with distinct DOK3 expression patterns

• Single-cell spatial profiling:

  • Multiplex immunofluorescence with DOK3 antibodies for spatial analysis of tissue sections

  • Digital spatial profiling (DSP) to quantify DOK3 in precisely defined tissue regions

  • Imaging mass cytometry for high-parameter spatial analysis of DOK3 in relation to tissue microenvironment

  • Correlate DOK3 spatial distribution with functional tissue domains

• Single-cell omics integration:

  • Combine protein (DOK3) and gene expression data through CITE-seq approaches

  • Link DOK3 protein levels to single-cell transcriptomics

  • Develop computational methods to integrate DOK3 protein data with transcriptomic clusters

  • Correlate DOK3 status with regulatory network analysis from single-cell data

These approaches parallel methods used in transplant rejection studies that integrate multiple biomarkers at single-cell resolution , providing methodological frameworks applicable to DOK3 research.

What are the considerations for developing phospho-specific DOK3 antibodies for signaling research?

Developing and validating phospho-specific DOK3 antibodies requires specialized approaches:

• Strategic epitope selection:

  • Identify functionally relevant phosphorylation sites through mass spectrometry

  • Focus on tyrosine residues known to be phosphorylated during signaling events

  • Consider sequence conservation across species if multi-species reactivity is desired

  • Evaluate surrounding amino acid context for antibody accessibility

• Validation requirements for phospho-antibodies:

  • Test with phosphatase-treated negative controls

  • Validate with phosphomimetic and non-phosphorylatable DOK3 mutants

  • Confirm specificity with competing phospho and non-phospho peptides

  • Demonstrate stimulus-dependent phosphorylation in appropriate cell models

• Application-specific considerations:

  Application	Special Considerations for Phospho-DOK3 Antibodies
  Western blot	Rapid sample processing to preserve phosphorylation
  Flow cytometry	Methanol permeabilization may better preserve phospho-epitopes
  IHC/IF	Test multiple fixation protocols to optimize epitope detection
  IP/ChIP	Validate retrieval of phosphorylated protein specifically

• Functional correlation strategies:

  • Correlate phospho-DOK3 detection with downstream signaling events

  • Use kinase inhibitors to confirm specificity of the phosphorylation event

  • Implement kinetic analyses to track phosphorylation dynamics

  • Develop multiplexed assays to simultaneously detect multiple phosphorylation sites

How can DOK3 antibodies contribute to understanding disease mechanisms in immunological disorders?

DOK3 antibodies can provide valuable insights into disease mechanisms through these research approaches:

• Clinical sample analysis:

  • Compare DOK3 expression/phosphorylation in patient vs. healthy control samples

  • Correlate DOK3 status with disease activity markers in autoimmune conditions

  • Perform longitudinal analysis during disease progression or treatment response

  • Integrate with genetic information (SNPs, mutations) affecting DOK3 function

• Mechanistic disease models:

  • Utilize DOK3 antibodies in animal models of autoimmunity, inflammation, or cancer

  • Track DOK3 regulation during disease development and therapeutic intervention

  • Study cell type-specific DOK3 function in complex disease environments

  • Investigate DOK3 as a biomarker for disease subtypes or treatment response

• Therapeutic target assessment:

  • Use DOK3 antibodies to monitor signaling pathway modulation by drug candidates

  • Develop assays for high-throughput screening of compounds affecting DOK3 pathways

  • Investigate DOK3 as a potential therapeutic target itself

  • Explore DOK3 status as a predictive biomarker for immunotherapy response

• Translational research applications:

  • Develop standardized DOK3 detection protocols for clinical research

  • Create tissue microarray-based screening for DOK3 in disease cohorts

  • Integrate DOK3 analysis into immune monitoring platforms

  • Establish DOK3 reference ranges in various tissue and cell types

Research on antibody-mediated rejection in kidney transplantation demonstrates how antibody markers can be integrated with molecular diagnostic systems to understand disease mechanisms , providing a translational research framework applicable to DOK3 studies.