DOK2 Antibody, FITC conjugated

What is DOK2 and why is it important in research?

DOK2 (Downstream of tyrosine kinase 2) is an enzymatically inert adaptor or scaffolding protein that provides a docking platform for the assembly of multimolecular signaling complexes . As a well-known tumor suppressor gene located on human chromosome 8p21.3, DOK2 acts through tyrosine kinase receptors including EGFR, PDGFR, and Her-2/NEU-8 via negative feedback modulation of protein tyrosine kinase signal transduction . This protein is involved in various cellular processes including proliferation, differentiation, and apoptosis, making it a significant target for research in cell biology, immunology, and cancer studies .

DOK2 plays important roles in:

• Modulating cellular proliferation induced by cytokines like IL-4, IL-2, and IL-3

• Attenuating EGF-stimulated MAP kinase activation

• Regulating T-cell receptor signaling and memory formation

• Potentially modulating Bcr-Abl signaling pathways

What is the difference between DOK2 antibodies and DOK2-FITC conjugated antibodies?

DOK2 antibodies refer to any antibodies that specifically recognize and bind to the DOK2 protein, while DOK2-FITC conjugated antibodies have been chemically linked to fluorescein isothiocyanate (FITC), a bright green fluorescent dye. The FITC conjugation allows direct visualization of the DOK2 protein in microscopy and flow cytometry applications without requiring secondary antibodies .

The practical difference lies in the methodology:

• Unconjugated DOK2 antibodies require a labeled secondary antibody or detection system in applications like Western blot and immunohistochemistry

• FITC-conjugated DOK2 antibodies enable direct detection via fluorescence microscopy or flow cytometry, simplifying experimental protocols and reducing background in multicolor experiments

What are the recommended applications for DOK2-FITC conjugated antibodies?

DOK2-FITC conjugated antibodies are particularly useful for:

• Immunofluorescence (IF) microscopy with recommended dilutions of 1:50-200

• Flow cytometry for detecting DOK2 expression in various cell populations

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) with recommended dilutions of 1:50-200

• Colocalization studies with other proteins labeled with different fluorophores

• Tracking DOK2 expression and localization in live or fixed cells

When using these antibodies, optimal results are achieved by first validating the antibody specificity in your experimental system and optimizing the antibody concentration for your specific application.

How should I prepare samples for optimal staining with DOK2-FITC antibodies?

For optimal staining with DOK2-FITC conjugated antibodies, sample preparation techniques depend on your application:

For IHC on paraffin-embedded tissues:

• Section tissues at 4-6 μm thickness and mount on positively charged slides

• Deparaffinize with xylene and rehydrate through an ethanol gradient

• Perform antigen retrieval using microwave heating with 10 mM Tris/EDTA buffer pH 9.0

• Block endogenous peroxidase activity using hydrogen peroxide

• Apply protein blocking solution to reduce non-specific binding

• Incubate with DOK2-FITC antibody at appropriate dilution (1:50-200)

• Wash thoroughly and proceed with nuclear counterstaining if desired

For immunofluorescence on cultured cells:

• Grow cells on coverslips or in chamber slides

• Fix with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Block with 1-5% BSA or normal serum for 30-60 minutes

• Incubate with DOK2-FITC antibody (1:100-1:200 dilution)

• Wash and counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

How can I validate the specificity of DOK2-FITC antibodies for my research?

Validating antibody specificity is crucial for reliable research outcomes. For DOK2-FITC antibodies, consider these approaches:

• Positive and negative controls:

  • Use cell lines known to express DOK2 (positive control) and those with low/no expression (negative control)

  • Consider using DOK2 knockout cells or tissues as definitive negative controls

• Western blot validation:

  • Perform Western blot with the unconjugated version of the same antibody clone

  • Confirm a single band at approximately 55 kDa, which is the expected molecular weight of DOK2

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Compare staining patterns between blocked and unblocked antibody

• siRNA knockdown verification:

  • Transfect cells with DOK2-specific siRNA and control siRNA

  • Confirm reduced signal in DOK2 knockdown samples compared to controls

• Cross-reactivity assessment:

  • Test the antibody against closely related proteins like DOK1 to ensure specificity

  • Verify reactivity with human and mouse samples as indicated in the product specifications

What controls should I include when using DOK2-FITC antibodies for immunofluorescence?

For robust immunofluorescence experiments with DOK2-FITC antibodies, include these essential controls:

• Isotype control: Use FITC-conjugated rabbit IgG at the same concentration as the DOK2-FITC antibody to assess non-specific binding

• Autofluorescence control: Include an unstained sample to determine background autofluorescence of your cells or tissues

• Single-stain controls: When performing multi-color immunofluorescence, include single-stained samples for each fluorophore to set compensation parameters

• Biological controls:

  • Positive control: Cells known to express DOK2 (e.g., immune cells, certain cancer cell lines)

  • Negative control: Cells with low/no DOK2 expression or DOK2 knockout cells

• Technical controls:

  • Secondary antibody-only control (when using indirect methods)

  • Blocking peptide control to demonstrate staining specificity

• Phosphorylation state controls: When studying phosphorylated DOK2 (e.g., at Tyr299), include samples treated with phosphatase inhibitors versus phosphatase-treated samples

How can DOK2-FITC antibodies be used to study DOK2's role in T-cell signaling and memory formation?

DOK2 plays a critical role in T-cell signaling and memory formation, making FITC-conjugated antibodies valuable tools for this research. Based on recent findings, here's a methodological approach:

• Experimental design for T-cell signaling studies:

  • Isolate CD8+ T cells from wild-type and DOK1/DOK2 double knockout (DKO) mice

  • Stimulate cells with CD3 mAbs to activate TCR signaling

  • Use DOK2-FITC antibodies in flow cytometry or microscopy to track changes in DOK2 expression and localization

  • Simultaneously assess phosphorylation levels of AKT and ERK1/2 as downstream markers of enhanced TCR signaling

• Memory T-cell differentiation analysis:

  • Pre-stimulate CD8+ T cells in vitro and monitor effector memory T-cell percentages

  • Use DOK2-FITC antibodies along with memory T-cell markers (CD44, CD62L) in multicolor flow cytometry

  • Analyze how DOK2 expression correlates with memory phenotype development

Research has shown that DOK1/DOK2 depletion in CD8+ T cells after in vitro pre-stimulation induces a higher percentage of effector memory T cells and upregulates TCR signaling cascade components, particularly phosphorylated AKT and ERK . This suggests DOK2 plays a regulatory role in memory T-cell formation, which can be effectively studied using fluorescently labeled antibodies.

What approaches can be used to investigate DOK2 phosphorylation states using phospho-specific and FITC-conjugated antibodies?

Investigating DOK2 phosphorylation states is crucial for understanding its function in signaling pathways. Here's a methodological approach combining phospho-specific and FITC-conjugated antibodies:

• Dual immunofluorescence strategy:

  • Use phospho-specific antibodies (e.g., anti-DOK2 phospho-Tyr299) for primary detection

  • Follow with FITC-conjugated secondary antibodies or directly conjugated phospho-DOK2-FITC antibodies

  • Counterstain with other cellular markers to assess colocalization of phosphorylated DOK2

• Stimulation-response experiments:

  • Treat cells with stimulants known to induce DOK2 phosphorylation (e.g., EGF, platelet-derived growth factors)

  • Fix cells at various time points (0, 5, 15, 30, 60 minutes)

  • Perform immunofluorescence with phospho-DOK2 antibodies

  • Quantify fluorescence intensity changes over time using image analysis software

• Pharmacological interventions:

  • Pre-treat cells with kinase inhibitors (e.g., Src family kinase inhibitors)

  • Stimulate with appropriate agonists

  • Assess DOK2 phosphorylation status using immunofluorescence or Western blot

  • Compare phosphorylation levels between treated and control samples

• Sample preparation for phospho-protein analysis:

  • Rapidly fix samples to preserve phosphorylation states

  • Include phosphatase inhibitors in all buffers

  • Perform microwave antigen retrieval with 10 mM Tris/EDTA buffer (pH 9.0) for tissue sections

How can I use DOK2-FITC antibodies to investigate DOK2's role as a tumor suppressor in cancer research?

DOK2 has been identified as a tumor suppressor, making it an important target for cancer research. Here's how to leverage DOK2-FITC antibodies in this context:

• Tumor tissue microarray analysis:

  • Create tissue microarrays containing multiple tumor samples and matched normal tissues

  • Perform immunofluorescence staining with DOK2-FITC antibodies

  • Quantify DOK2 expression levels across different cancer types and stages

  • Correlate expression patterns with clinical outcomes and pathological features

• Functional studies in cancer cell lines:

  • Manipulate DOK2 expression through overexpression or knockdown approaches

  • Assess changes in:

    • Cell proliferation (using proliferation markers alongside DOK2-FITC staining)

    • Ras-Raf-MAPK and PI3K-Akt pathway activation

    • Response to EGF stimulation and EGFR inhibition

  • Use DOK2-FITC antibodies to confirm expression changes and study subcellular localization

• Signaling pathway analysis:

  • Investigate DOK2's role in the negative regulation of Ras signaling in the Ras-Raf-MAPK pathway

  • Study how DOK2 inhibits Akt phosphorylation through the PI3K-Akt pathway

  • Use dual staining approaches with DOK2-FITC and antibodies against pathway components

• In vivo tumor models:

  • Develop xenograft models using DOK2-manipulated cancer cells

  • Harvest tumors for immunofluorescence analysis with DOK2-FITC antibodies

  • Correlate DOK2 expression with tumor growth kinetics and metastatic potential

What are common issues when using FITC-conjugated antibodies and how can they be addressed?

When working with DOK2-FITC conjugated antibodies, researchers may encounter several challenges:

• Photobleaching:

  • Issue: FITC is prone to photobleaching during microscopy

  • Solution: Minimize exposure to light; use anti-fade mounting media containing agents like DABCO or PPD; consider using LED light sources instead of mercury lamps; capture FITC images first in multi-channel imaging

• Autofluorescence:

  • Issue: Tissues may have natural fluorescence in the FITC channel

  • Solution: Use autofluorescence quenching agents like Sudan Black B (0.1-0.3%); include unstained controls; consider using spectral unmixing on confocal microscopes

• pH sensitivity:

  • Issue: FITC fluorescence is optimal at alkaline pH (>7.5) and decreases at lower pH

  • Solution: Ensure buffers are properly prepared and at optimal pH; avoid acidic mounting media

• Low signal-to-noise ratio:

  • Issue: High background with weak specific signal

  • Solution: Optimize antibody concentration; increase blocking time/concentration; use more extensive washing steps; consider alternative fixation methods

• Cross-reactivity:

  • Issue: Non-specific binding to other proteins

  • Solution: Validate antibody specificity using techniques described in FAQ 2.2; increase blocking time; consider using different blocking agents (BSA, normal serum, commercial blockers)

How can I optimize staining protocols for different sample types when using DOK2-FITC antibodies?

Optimizing staining protocols for different sample types requires systematic adjustment of multiple parameters:

For cell lines:

• Fixation: Test different fixatives (4% PFA, methanol, acetone) and fixation times

• Permeabilization: Optimize detergent type (Triton X-100, saponin) and concentration (0.1-0.5%)

• Blocking: Test different blocking agents (1-5% BSA, normal serum, commercial blockers)

• Antibody dilution: Test a range of dilutions around the recommended 1:100-1:200

• Incubation conditions: Compare room temperature vs. 4°C, and different incubation times

For tissue sections:

• Antigen retrieval: Compare heat-induced epitope retrieval methods:

  • Microwave heating with 10 mM Tris/EDTA buffer pH 9.0

  • Pressure cooker with citrate buffer pH 6.0

  • Enzymatic retrieval with proteinase K

• Section thickness: Optimize between 4-6 μm for paraffin sections

• Antibody penetration: Increase incubation time for thicker sections

• Background reduction: Test Sudan Black B treatment to reduce autofluorescence

• Signal amplification: Consider tyramide signal amplification if signal is weak

For flow cytometry:

• Cell preparation: Optimize fixation and permeabilization for intracellular staining

• Antibody concentration: Titrate to determine optimal staining index

• Buffer composition: Test different staining buffers with/without serum

• Compensation: Properly compensate for spectral overlap when using multiple fluorophores

• Gating strategy: Develop appropriate gating based on controls

How does phosphorylation status affect DOK2 antibody binding, and what methodological approaches can address this?

The phosphorylation status of DOK2 can significantly impact antibody binding, particularly when using antibodies targeting specific epitopes:

• Effect of phosphorylation on epitope accessibility:

  • Phosphorylation can induce conformational changes that may mask or expose certain epitopes

  • This can affect binding efficiency of antibodies not specifically designed to recognize phosphorylated sites

  • Solution: Use a combination of phospho-specific and total DOK2 antibodies to get a complete picture

• Methodological approaches:

  • Phosphatase treatment controls: Treat duplicate samples with lambda phosphatase to remove phosphate groups

  • Kinase treatment: Enhance phosphorylation using appropriate kinase activators (e.g., EGF treatment for DOK2 Tyr299 phosphorylation)

  • Comparison of phospho-specific vs. total DOK2 antibodies: Use both in parallel experiments

  • Site-directed mutagenesis: Create phospho-mimetic (e.g., Y299D) or phospho-null (e.g., Y299F) DOK2 mutants to validate antibody specificity

• Optimizing detection of phosphorylated DOK2:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all buffers

  • Process samples quickly to minimize dephosphorylation

  • Consider using phospho-specific antibodies like anti-DOK2 (phospho Tyr299)

  • Use dual staining with total DOK2-FITC and phospho-DOK2 antibodies detected with a different fluorophore

• Sample preparation considerations:

  • Different fixation methods can differentially preserve phospho-epitopes

  • Formalin fixation may better preserve phosphorylated residues compared to alcohol-based fixatives

  • Antigen retrieval conditions may need to be optimized specifically for phospho-epitopes

How can DOK2-FITC antibody data be integrated with other molecular techniques to study DOK2's role in signaling networks?

Integrating DOK2-FITC antibody data with other molecular techniques provides a comprehensive understanding of DOK2's role in signaling networks:

• Multi-omics integration approach:

  • Combine immunofluorescence data with phosphoproteomics

  • Correlate DOK2 localization/expression with RNA-seq transcriptome data

  • Integrate with interactome data from proximity labeling or co-immunoprecipitation studies

• Methodological workflow:

  • Use DOK2-FITC antibodies for cellular localization and expression level analysis

  • Perform parallel phosphoproteomics to identify phosphorylation changes in MAPK and Akt pathways

  • Conduct ChIP-seq to identify transcriptional changes downstream of DOK2 signaling

  • Analyze data using pathway analysis tools to identify signaling networks

• Single-cell multi-parameter analysis:

  • Combine DOK2-FITC flow cytometry with phospho-flow for ERK and Akt

  • Assess correlation between DOK2 expression and downstream signaling at single-cell level

  • Use dimensionality reduction techniques (t-SNE, UMAP) to visualize multi-parameter data

• Temporal dynamics analysis:

  • Use live-cell imaging with DOK2-FITC antibodies (if cell-permeable versions are available)

  • Track DOK2 localization changes following stimulation

  • Correlate with real-time biosensors for MAPK or Akt activity

What are the latest research findings on DOK2's role in immune cell function that can be studied using FITC-conjugated antibodies?

Recent research has revealed critical roles for DOK2 in immune cell function that can be further investigated using FITC-conjugated antibodies:

• T-cell memory formation and signaling:

  • Recent finding: DOK1/DOK2 depletion in CD8+ T cells induces higher percentages of effector memory T cells and upregulates TCR signaling cascade components

  • Application: Use DOK2-FITC antibodies in flow cytometry panels with memory markers (CD44/CD62L) to track DOK2 expression during memory T-cell differentiation

• Negative regulation of CD8+ T-cell responses:

  • Recent finding: DOK1 and DOK2 negatively regulate the overactivation of CD8+ T-cells and promote memory cell formation

  • Application: Study DOK2 expression and localization during viral antigen stimulation using immunofluorescence microscopy

• Platelet function regulation:

  • Recent finding: DOK2 plays an important role in integrin-induced signal transduction and forms immune complexes with integrin αIIβ3

  • Application: Use DOK2-FITC antibodies to study colocalization with integrins in platelets using confocal microscopy

• T-cell receptor signaling regulation:

  • Recent finding: DOK2 functions as a negative regulator of T-cell receptor signaling by inhibiting tyrosine phosphorylation and the Ras signaling pathway

  • Application: Combine DOK2-FITC staining with phospho-specific antibodies against TCR signaling components in stimulated T cells

• CD200R-mediated inhibitory signaling:

  • Recent finding: CD200R directly recruits DOK2 and activates RasGAP, inhibiting bone marrow cell activation

  • Application: Use dual-color immunofluorescence with DOK2-FITC and CD200R antibodies to study their interaction

How can researchers quantitatively analyze DOK2 expression patterns in different cell types and disease states using FITC-conjugated antibodies?

Quantitative analysis of DOK2 expression patterns using FITC-conjugated antibodies requires rigorous methodological approaches:

• Flow cytometry-based quantification:

  • Method: Create a standardized flow cytometry panel including DOK2-FITC

  • Analysis: Calculate median fluorescence intensity (MFI) and percent positive cells

  • Quantification: Use calibration beads with known fluorophore molecules to convert MFI to molecules of equivalent soluble fluorochrome (MESF)

  • Application: Compare DOK2 expression levels across different immune cell subsets or disease states

• Quantitative microscopy approaches:

  • Method: Standardize image acquisition settings (exposure time, gain, laser power)

  • Analysis tools: Use software like ImageJ, CellProfiler, or QuPath for automated quantification

  • Parameters to measure:

    • Mean fluorescence intensity per cell

    • Nuclear-to-cytoplasmic ratio of DOK2 expression

    • Colocalization coefficients with signaling partners

  • Normalization: Include internal controls in each experiment

• Tissue microarray analysis for disease states:

  • Method: Create tissue microarrays with multiple samples from different disease states

  • Staining: Use DOK2-FITC antibodies with standardized protocols

  • Quantification: Apply automated image analysis algorithms to quantify expression levels

  • Statistical analysis: Correlate DOK2 expression with clinical parameters and outcomes

• Single-cell analysis workflow:

  • Method: Combine DOK2-FITC antibody staining with single-cell sequencing approaches

  • Analysis: Correlate protein expression with transcriptomic profiles

  • Application: Identify cell subpopulations with distinct DOK2 expression patterns

  • Visualization: Use t-SNE or UMAP plots to display multidimensional data

What are emerging applications for DOK2-FITC antibodies in cancer immunotherapy research?

As cancer immunotherapy continues to evolve, DOK2-FITC antibodies offer valuable tools for several emerging research directions:

• Predictive biomarker development:

  • Given DOK2's role as a tumor suppressor , its expression levels could serve as biomarkers for immunotherapy response

  • Method: Use DOK2-FITC antibodies to screen patient samples prior to immunotherapy

  • Application: Correlate DOK2 expression patterns with clinical outcomes

  • Potential: Develop companion diagnostics for immunotherapy patient selection

• T-cell engineering enhancement:

  • Recent finding: Targeting TCR-signaling inhibitory proteins like CISH improves TCR activation and tumor clearing

  • Application: Investigate DOK2 manipulation in adoptive T-cell therapies

  • Method: Use DOK2-FITC antibodies to monitor expression in engineered T cells

  • Potential: Enhance CAR-T or TCR-T cell therapy efficacy by modulating DOK2 levels

• Combination therapy optimization:

  • Rationale: DOK2's role in modulating signaling pathways suggests potential synergies with targeted therapies

  • Method: Use DOK2-FITC antibodies to monitor expression changes during combination treatments

  • Application: Study how DOK2 expression affects response to immune checkpoint inhibitors

  • Potential: Identify rational drug combinations based on DOK2 expression patterns

• Tumor microenvironment evaluation:

  • Method: Multiplex immunofluorescence with DOK2-FITC and other immune cell markers

  • Application: Characterize DOK2 expression in tumor-infiltrating lymphocytes

  • Analysis: Correlate spatial distribution of DOK2+ cells with therapeutic outcomes

  • Potential: Discover new prognostic indicators and therapeutic targets

What methodological advances might improve the use of DOK2-FITC antibodies in future research?

Several methodological advances could enhance the utility of DOK2-FITC antibodies in future research:

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize DOK2 nanoscale organization in signaling clusters

  • Light-sheet microscopy for rapid 3D imaging of DOK2 distribution in tissue samples

  • Multiphoton microscopy for deep tissue imaging of DOK2 in intact organs

  • FRET-based approaches to study DOK2 interactions with binding partners

• Single-molecule approaches:

  • Single-molecule tracking of DOK2 dynamics using brighter and more stable fluorophores

  • Expansion microscopy to physically enlarge specimens for improved resolution

  • Correlative light and electron microscopy to link DOK2 localization with ultrastructure

• Multiplex imaging enhancements:

  • Cyclic immunofluorescence to analyze DOK2 alongside dozens of other markers

  • Mass cytometry (CyTOF) with metal-tagged DOK2 antibodies for high-parameter analysis

  • Spatial transcriptomics combined with DOK2 protein detection

  • Quantum dot-conjugated antibodies for improved stability and brightness

• Live-cell applications:

  • Development of cell-permeable DOK2-FITC nanobodies for live-cell imaging

  • Optogenetic tools to manipulate DOK2 function while monitoring localization

  • Biosensors to detect DOK2 conformational changes upon phosphorylation

• Computational methods:

  • Machine learning algorithms for automated DOK2 expression pattern recognition

  • Integrative multi-omics approaches linking DOK2 protein expression with genetic and transcriptomic data

  • Digital pathology workflows for large-scale analysis of DOK2 expression in clinical samples