DOT2 Antibody

What is DOK2 and what is its biological significance?

DOK2 (Docking protein 2) is an enzymatically inert adaptor or scaffolding protein that provides a docking platform for the assembly of multimolecular signaling complexes. Functionally, DOK2 may modulate cellular proliferation induced by several interleukins, including IL-4, IL-2, and IL-3. It may also be involved in modulating Bcr-Abl signaling and attenuates EGF-stimulated MAP kinase activation. These properties make DOK2 an important research target in cellular signaling studies, particularly in immune cell function and cancer research contexts .

What is the difference between DOT2 software and DOK2 antibodies?

While similarly named, these represent entirely different scientific tools. DOT2 is a computational software suite designed for macromolecular docking that provides automated construction of improved biophysical models based on molecular coordinates. It employs convolution-based rigid-body docking algorithms to predict protein-protein interactions . In contrast, DOK2 antibodies are immunological reagents designed to bind specifically to Docking protein 2 (DOK2), enabling its detection and study in biological samples. Researchers should be careful not to confuse these distinct scientific resources despite their similar nomenclature .

What applications are DOK2 antibodies commonly used for?

DOK2 antibodies are primarily employed in several key experimental applications:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) for localization studies

• Immunocytochemistry/Immunofluorescence (ICC/IF) for subcellular localization

These applications allow researchers to investigate DOK2 expression patterns, protein-protein interactions, and signaling pathway involvement. The antibodies are particularly useful for studying DOK2's role in modulating cellular proliferation and signal transduction pathways .

What is a Dot-immunobinding assay and how does it relate to antibody research?

The Dot-immunobinding assay (Dot-Iba) is a simple, reproducible immunodiagnostic method where antibodies or antigens are directly applied ("dotted") onto nitrocellulose membrane discs. The presence of antigen-antibody complexes is visualized using enzyme-conjugated antiglobulins and substrates, with positive results indicated by a purple-pink colored, insoluble product. This assay is advantageous because it allows processing of multiple specimens simultaneously, requires only 4-6 hours to complete, and involves simpler technical steps than other immunoassays like ELISA. Dot-Iba has shown high specificity (no false positives) with moderate sensitivity (60% for tuberculous meningitis diagnosis), making it particularly suitable for laboratories with limited resources .

How do I select the appropriate DOK2 antibody for my experimental needs?

Selecting the appropriate DOK2 antibody requires consideration of several factors:

• Experimental application (WB, IHC-P, ICC/IF)

• Species reactivity (human, mouse, etc.)

• Antibody type (monoclonal vs. polyclonal)

• Epitope recognition region (N-terminal, C-terminal, or internal)

• Validation status for your specific application

For instance, commercially available antibodies like those from Abcam (ab131488) are rabbit polyclonal antibodies that recognize epitopes within amino acids 250-350 of human DOK2 and have been validated for Western blot, immunohistochemistry, and immunofluorescence applications with human samples .

How can DOK2 antibodies be validated for specificity in research applications?

Validating DOK2 antibodies for specificity involves multiple complementary approaches:

• Western blot analysis: Observe a single band at the expected molecular weight (~45 kDa for DOK2)

• siRNA knockdown: Compare antibody signal in normal cells versus cells with DOK2 expression reduced by siRNA treatment

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide to demonstrate signal reduction

• Cross-reactivity testing: Test against related DOK family proteins to ensure specificity

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm consistent results

For highest confidence, validation should include positive controls (cell lines known to express DOK2, such as Jurkat cells) and negative controls (cell lines with minimal DOK2 expression or knockdown models) .

What are the best practices for conjugating DOK2 antibodies for advanced imaging techniques?

Conjugating DOK2 antibodies for advanced imaging requires careful consideration of the following factors:

• Antibody purity: Ensure high purity (>95%) before conjugation to maximize efficiency

• Buffer optimization: Remove amine-containing buffers (Tris) and switch to PBS or bicarbonate buffer

• Conjugation ratio: Optimize fluorophore-to-antibody ratio (typically 4-8 molecules per antibody)

• Reaction conditions: Control pH (7.0-8.5), temperature (room temperature), and duration (1-4 hours)

• Purification: Remove unbound fluorophores using size exclusion chromatography

The methodology used for DOTA conjugation to rituximab provides a useful model. In this approach, a stock solution of the conjugation agent is prepared, and the conjugation reaction is carefully controlled. After conjugation, the number of conjugated molecules per antibody can be determined spectrophotometrically, aiming for approximately 3-5 molecules per antibody to maintain immunoreactivity while providing sufficient signal .

How does DOT2 computational docking complement antibody-based research?

DOT2 computational docking provides a valuable complement to antibody-based research through:

• Epitope prediction: Identifying potential antibody binding sites on DOK2

• Structural insights: Predicting how antibody binding affects DOK2's interaction with binding partners

• Rational design: Guiding the development of antibodies with improved specificity and affinity

• Mechanism elucidation: Understanding how DOK2 functions in signaling complexes

The DOT2 software employs a three-step process: preprocessing to calculate electrostatic and van der Waals properties, docking using grid-based calculations, and evaluation. Using standard parameters, DOT2 can evaluate approximately 108 billion possible configurations between molecules, enabling comprehensive exploration of potential interaction interfaces .

What factors affect the immunoreactivity of conjugated antibodies like DOK2?

Several key factors influence the immunoreactivity of conjugated antibodies:

In studies with DOTA-conjugated antibodies, optimal conditions yielded immunoreactivity of approximately 70%, indicating that about 70% of the conjugated antibody remained capable of binding to its target. Higher conjugation ratios typically result in lower immunoreactivity, demonstrating the importance of optimizing the conjugation process .

How can DOK2 antibodies be employed in multiplexed assays for signaling pathway analysis?

DOK2 antibodies can be integrated into multiplexed signaling pathway analysis through:

• Multi-color immunofluorescence: Using spectrally distinct fluorophores to simultaneously detect DOK2 and interacting proteins

• Phospho-specific antibody panels: Combining DOK2 antibodies with phospho-specific antibodies targeting downstream effectors

• Proximity ligation assays: Detecting protein-protein interactions involving DOK2 with single-molecule resolution

• Mass cytometry (CyTOF): Labeling antibodies with distinct metal isotopes for highly multiplexed analyses

• Sequential immunodetection: Stripping and reprobing membranes with multiple antibodies

When designing such assays, antibody compatibility must be considered to avoid cross-reactivity and ensure proper epitope accessibility. For optimal results, validation should include appropriate controls and careful optimization of antibody concentrations .

What is the optimal protocol for Western blotting using DOK2 antibodies?

The optimal Western blotting protocol for DOK2 antibodies includes:

• Sample preparation: Lyse cells in RIPA buffer with protease/phosphatase inhibitors

• Protein separation: Run 20-30 μg protein on 10-12% SDS-PAGE

• Transfer: Use PVDF membrane (0.45 μm) for optimal protein binding

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute DOK2 antibody 1:500 in blocking buffer; incubate overnight at 4°C

• Washing: Wash 3-4 times with TBST, 5 minutes each

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000; incubate for 1 hour

• Detection: Develop using enhanced chemiluminescence

The expected band size for DOK2 is approximately 45 kDa. Jurkat cell extracts serve as positive controls based on validated antibody testing .

How should immunohistochemistry protocols be optimized for DOK2 detection in tissue samples?

Optimizing immunohistochemistry for DOK2 detection requires:

• Tissue fixation: Use 10% neutral buffered formalin; limit fixation time to 24 hours

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Peroxidase blocking: Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Protein blocking: Block with 5-10% normal serum in PBS for 30-60 minutes

• Primary antibody: Dilute DOK2 antibody 1:100 in blocking buffer; incubate overnight at 4°C

• Detection system: Use polymer-based detection systems for enhanced sensitivity

• Counterstaining: Lightly counterstain with hematoxylin to maintain signal visibility

• Controls: Include positive control tissues and negative controls (primary antibody omitted)

These parameters should be adjusted based on tissue type and fixation conditions. For paraffin-embedded human tissues, commercial DOK2 antibodies have been validated at 1:100 dilution with appropriate antigen retrieval .

What are the key considerations for using DOK2 antibodies in flow cytometry?

When using DOK2 antibodies for flow cytometry, researchers should consider:

• Cell preparation: Use fresh cells or properly fixed samples to maintain antigen integrity

• Permeabilization: Since DOK2 is intracellular, use appropriate permeabilization (0.1% Triton X-100 or commercial permeabilization buffers)

• Antibody concentration: Titrate antibody to determine optimal concentration (typically starting at 1:50-1:100)

• Incubation conditions: Incubate for 30-45 minutes at 4°C in the dark

• Washing steps: Perform 2-3 washes with PBS containing 1% BSA

• Secondary antibody: Use fluorophore-conjugated secondary antibodies appropriate for your cytometer configuration

• Controls: Include unstained, secondary-only, and isotype controls

For evaluating immunoreactivity, protocols similar to those used for other antibodies can be applied. For example, the immunoreactivity of DOTA-conjugated antibodies has been assessed by incubating CD20-positive Raji cells with the antibody, followed by analysis with flow cytometry .

How can Dot-immunobinding assay be optimized for DOK2 detection?

Optimizing Dot-immunobinding assay for DOK2 detection involves:

• Membrane preparation: Use nitrocellulose membrane discs with 0.45 μm pore size

• Sample application: Apply 2-5 μL of purified DOK2 protein or cell lysate directly onto membrane

• Drying: Allow spots to dry completely (15-30 minutes at room temperature)

• Blocking: Block with 5% BSA in TBST for 1 hour to reduce background

• Primary antibody: Apply DOK2 antibody diluted in blocking buffer (1:500-1:1000)

• Washing: Wash membranes 3-4 times with TBST

• Detection: Use enzyme-conjugated secondary antibody and appropriate substrate

• Result assessment: Positive results appear as purple-pink colored dots

This approach allows for processing multiple samples simultaneously, with the entire procedure requiring only 4-6 hours. The simplicity of this method makes it particularly attractive for rapid screening or laboratories with limited resources .

What quality control measures should be implemented when using DOK2 antibodies in research?

Implementing robust quality control for DOK2 antibody-based research requires:

• Antibody validation: Confirm specificity through Western blot, showing a single band at ~45 kDa

• Lot testing: Test each new antibody lot against a reference lot to ensure consistent performance

• Positive controls: Include known DOK2-expressing samples (e.g., Jurkat cells) in each experiment

• Negative controls: Use primary antibody omission and irrelevant isotype controls

• Cross-reactivity assessment: Test for potential cross-reactivity with other DOK family proteins

• Signal quantification: Include standard curves when performing quantitative analyses

• Reproducibility testing: Perform technical and biological replicates to ensure consistency

Following stringent antibody validation approaches, as described by commercial suppliers like Diagenode, can significantly enhance confidence in experimental results. These approaches include testing antibodies against peptide arrays and using siRNA knockdown to confirm specificity .

How should researchers address weak or absent signal when using DOK2 antibodies?

When facing weak or absent signals with DOK2 antibodies, consider these troubleshooting steps:

• Expression levels: Confirm DOK2 expression in your sample using RT-PCR

• Antibody concentration: Increase primary antibody concentration (e.g., from 1:500 to 1:250)

• Incubation time: Extend primary antibody incubation (overnight at 4°C)

• Antigen retrieval: Optimize antigen retrieval methods for fixed tissues

• Detection system: Switch to more sensitive detection systems

• Sample handling: Check for protein degradation in samples

• Epitope accessibility: Try antibodies targeting different DOK2 epitopes

• Signal amplification: Implement tyramide signal amplification or similar methods

For Western blotting specifically, loading more protein (30-50 μg) and using fresh transfer buffers can improve results. For IHC and ICC, extending development time and using polymer-based detection systems may enhance sensitivity .

What strategies can address non-specific binding when using DOK2 antibodies?

To reduce non-specific binding with DOK2 antibodies:

• Blocking optimization: Extend blocking time or try alternative blocking agents

• Antibody dilution: Further dilute primary antibody to reduce non-specific interactions

• Washing stringency: Increase washing duration and buffer stringency (add 0.1-0.3% Triton X-100)

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies

• Pre-adsorption: Pre-adsorb antibody with cell/tissue lysates from negative control samples

• Buffer optimization: Adjust salt concentration in washing and incubation buffers

• Control experiments: Perform peptide competition assays to confirm specificity

Non-specific binding can also be assessed through dot blot assays similar to those used for histone modification antibodies, where specificity is confirmed when the signal for the target peptide represents >70% of the total signal. Most high-quality antibodies exceed 90% specificity in such tests .

How can researchers distinguish between DOK2 and other DOK family proteins in their experiments?

Distinguishing between DOK2 and other DOK family proteins requires:

• Epitope selection: Choose antibodies targeting unique regions not conserved among DOK proteins

• Western blot analysis: Compare band patterns with predicted molecular weights (DOK2: ~45 kDa)

• Peptide arrays: Test antibody reactivity against peptide arrays of all DOK family members

• Knockout/knockdown controls: Use genetic approaches to specifically deplete DOK2

• Immunoprecipitation-mass spectrometry: Confirm protein identity through mass spectrometry

• Cross-reactivity testing: Systematically test antibody against recombinant DOK family proteins

When developing antibodies, immunization strategies similar to those used by companies like Diagenode can enhance specificity. These approaches include designing peptides to evoke maximal immune response, immunizing multiple animals, and extensive testing through ELISA, Western blot, and dot blot to ensure specificity .

What are the best practices for quantifying DOK2 expression in Western blots and immunostaining?

Quantifying DOK2 expression accurately requires:

• Loading controls: Use housekeeping proteins (β-actin, GAPDH) or total protein stains (Ponceau S)

• Linear dynamic range: Ensure detection is within the linear range of your imaging system

• Replicate analysis: Perform at least three biological replicates for statistical validity

• Normalization: Always normalize DOK2 signal to appropriate loading controls

• Software analysis: Use dedicated analysis software with background subtraction

• Standard curves: Include recombinant DOK2 standard curves for absolute quantification

• Batch processing: Process all comparable samples in the same experiment

For immunostaining quantification, consistent exposure settings, random field selection, and automated analysis algorithms help reduce bias. Present data as fold-change relative to control conditions rather than absolute values to account for inter-experimental variability .

How can computational approaches like DOT2 inform antibody-antigen interaction studies?

Computational approaches like DOT2 can enhance antibody-antigen studies through:

• Epitope mapping: Predicting antibody binding sites on DOK2 structure

• Binding energy estimation: Calculating theoretical binding affinities

• Conformational analysis: Predicting effects of mutations on epitope structure

• Cross-reactivity prediction: Identifying potential cross-reactive proteins

• Rational antibody design: Guiding development of improved antibodies

DOT2 software systematically translates and rotates one molecule around another, calculating interaction energies by convolving the potential field of the stationary molecule with atom-based properties of the moving molecule. With standard parameters (54,000 orientations and 1 Å grid spacing), DOT2 can evaluate approximately 108 billion configurations between molecules, providing comprehensive analysis of potential binding modes .

How can DOK2 antibodies be adapted for super-resolution microscopy techniques?

Adapting DOK2 antibodies for super-resolution microscopy involves:

• Conjugation optimization: Use bright, photostable fluorophores suitable for super-resolution

• Labeling density: Optimize antibody concentration for appropriate labeling density

• Sample preparation: Implement specialized fixation and mounting for super-resolution

• Secondary antibody strategy: Consider using F(ab) fragments to reduce linkage error

• Validation: Confirm specificity using co-localization with known interaction partners

For techniques like STORM or PALM, direct conjugation with photoactivatable/photoswitchable fluorophores may be necessary. The conjugation approaches developed for DOTA-rituximab can be adapted, ensuring that 3-4 fluorophores per antibody are attached while maintaining approximately 70% immunoreactivity .

What approaches can be used to study dynamic DOK2 interactions in living cells?

Studying dynamic DOK2 interactions in living cells requires:

• Genetic tagging: Generate DOK2-fluorescent protein fusions (GFP, mCherry)

• Nanobody development: Develop anti-DOK2 nanobodies for live-cell labeling

• FRET/BRET assays: Design fusion constructs to monitor protein-protein interactions

• Photoactivation experiments: Use photoactivatable DOK2 constructs to track protein movement

• Correlation spectroscopy: Apply fluorescence correlation spectroscopy to measure diffusion rates

These approaches complement traditional antibody-based methods and provide temporal information about DOK2 dynamics. The molecular docking predictions from DOT2 can guide the design of fusion constructs by identifying regions where tags would minimally interfere with interaction interfaces .

How can DOK2 antibodies be integrated with mass spectrometry for comprehensive signaling analysis?

Integrating DOK2 antibodies with mass spectrometry involves:

• Immunoprecipitation-MS: Use DOK2 antibodies to enrich protein complexes for MS analysis

• Proximity labeling: Combine DOK2 antibodies with BioID or APEX2 proximity labeling

• Phosphoproteomics: Use phospho-specific DOK2 antibodies to track signaling states

• Cross-linking MS: Apply cross-linking reagents before immunoprecipitation

• Absolute quantification: Develop DOK2 peptide standards for absolute quantification

These integrative approaches provide comprehensive views of DOK2-mediated signaling networks. The methodology used for DOTA-rituximab conjugation and characterization provides a useful model for preserving antibody functionality during these complex workflows .

What are the emerging applications of DOK2 antibodies in single-cell analysis techniques?

Emerging single-cell applications for DOK2 antibodies include:

• Single-cell Western blotting: Miniaturized Western blot for individual cells

• Mass cytometry: Metal-conjugated antibodies for highly multiplexed analysis

• Imaging mass cytometry: Spatial resolution of protein expression in tissues

• Microfluidic antibody capture: Capturing secreted proteins from single cells

• Single-cell proteomics: Integrating antibody-based detection with MS

These techniques enable unprecedented resolution of DOK2 expression heterogeneity and co-expression patterns. For optimal results, antibodies must be highly specific, as demonstrated through rigorous validation methods including peptide competition assays and knockout controls .

How might computational docking tools like DOT2 evolve to better support antibody research?

Future developments in computational docking tools may include:

• Integrated AI approaches: Machine learning models trained on antibody-antigen structures

• Flexibility modeling: Improved modeling of flexible regions in antibodies and antigens

• Epitope prediction: Advanced algorithms for B-cell epitope prediction

• Affinity estimation: More accurate binding affinity predictions

• Cloud-based platforms: Accessible interfaces for non-computational scientists

Current tools like DOT2 already offer significant capabilities, including grid-based calculations that can evaluate approximately 108 billion configurations between molecules. Future developments will likely focus on improving accuracy and incorporating experimental data to refine predictions .