ADO3 Antibody

What is DOK3 protein and what cellular functions does it regulate?

DOK3 (Docking protein 3) is an enzymatically inert adaptor or scaffolding protein that provides a docking platform for the assembly of multimolecular signaling complexes. It functions as a negative regulator of JNK signaling in B-cells through interaction with INPP5D/SHIP1 and may modulate ABL1 function. These interactions inhibit excessive activation signals, allowing DOK3 to maintain immune system homeostasis and ensure proper immune function . As a member of the DOK protein family, it plays a crucial role in regulating immune responses by facilitating protein-protein interactions that modulate signaling cascades following receptor activation.

What applications is the DOK3 antibody suitable for in laboratory research?

The DOK3 antibody (such as ab236609) has been validated for multiple laboratory applications, including Western Blotting (WB), Immunohistochemistry on paraffin-embedded sections (IHC-P), and Immunocytochemistry/Immunofluorescence (ICC/IF) . These diverse applications make it versatile for researchers investigating DOK3 protein expression and localization across different experimental contexts. The antibody has been successfully used to detect DOK3 in various tissue samples including heart, placenta, and tonsil tissue, demonstrating its utility across different biological systems .

What species reactivity does DOK3 antibody demonstrate?

Current DOK3 antibodies, such as the rabbit polyclonal antibody referenced in the search results, show confirmed reactivity with both mouse and human samples . This cross-species reactivity is particularly valuable for translational research that aims to correlate findings between animal models and human systems. When selecting a DOK3 antibody for research, it's important to verify species reactivity based on the specific experimental design and target tissue or cell type.

What are the optimal protocols for DOK3 antibody application in Western blotting?

For Western blotting applications, DOK3 antibody should be used at a dilution of approximately 1/500. The documented protocol shows successful detection in mouse heart and placenta tissue lysates using this dilution . The predicted band size for DOK3 is approximately 53 kDa. For optimal results, secondary detection should use an anti-rabbit IgG (such as goat polyclonal to rabbit IgG) at an appropriate dilution (1/50000 has been successful in published protocols) . Standard Western blotting procedures including proper protein extraction, SDS-PAGE separation, and transfer to membrane should be followed, with blocking in appropriate blocking buffer before antibody incubation.

What are the recommended protocols for immunohistochemical detection of DOK3?

For immunohistochemistry on paraffin-embedded tissue sections, a dilution of 1/100 has been shown to be effective for DOK3 detection . The protocol should include proper deparaffinization, antigen retrieval (which may vary depending on tissue type), blocking of endogenous peroxidases and non-specific binding sites, followed by primary antibody incubation (DOK3 antibody), appropriate secondary antibody application, and visualization using a suitable detection system. Successful staining has been documented in human tonsil tissue using this approach . Optimization may be required for different tissue types or experimental conditions.

How should researchers validate DOK3 antibody specificity for their particular application?

Antibody validation is a critical step to ensure experimental reliability. For DOK3 antibody, validation should include multiple complementary approaches:

• Positive and negative control samples (tissues/cells known to express or not express DOK3)

• Comparison with alternative antibodies targeting different epitopes of the same protein

• Knockdown or knockout validation where feasible

• Assessment of expected molecular weight in Western blot applications

• Comparison of staining patterns with published literature

Similar to validation approaches used for other antibodies, researchers might consider using domain-specific constructs to verify binding specificity, as demonstrated in the anti-idiotypic antibody characterization described in the literature .

How can DOK3 antibody be used to investigate immune system homeostasis mechanisms?

DOK3 antibody serves as a valuable tool for investigating the molecular mechanisms underlying immune system homeostasis. Researchers can design experiments to:

• Assess DOK3 expression levels in various immune cell populations under different activation states

• Investigate co-localization with binding partners like INPP5D/SHIP1 using dual immunofluorescence

• Examine changes in DOK3 expression or localization in models of immune dysfunction

• Study phosphorylation states of DOK3 following receptor activation

These approaches can help elucidate how DOK3 functions as a negative regulator of immune signaling cascades. The antibody can be used in combination with functional assays to correlate DOK3 expression or modification with changes in cellular responses to various stimuli.

What role does DOK3 play in B-cell signaling, and how can DOK3 antibody help elucidate these pathways?

DOK3 functions as a negative regulator of JNK signaling in B-cells through its interaction with INPP5D/SHIP1 . To investigate these pathways, researchers can employ DOK3 antibody in:

• Immunoprecipitation experiments to pull down DOK3 and identify interaction partners

• Phospho-specific Western blotting to track signaling cascade activation in the presence/absence of DOK3

• Immunocytochemistry to monitor subcellular localization changes during B-cell activation

• Flow cytometry to correlate DOK3 expression with B-cell maturation states or activation markers

These methodological approaches can reveal the spatiotemporal dynamics of DOK3 function within B-cell signaling networks and help identify novel regulatory mechanisms that maintain appropriate immune responses.

How do emerging antibody discovery technologies relate to improving research antibodies like DOK3 antibody?

Recent advances in antibody discovery could significantly improve research antibodies, including those targeting DOK3. Vanderbilt University Medical Center's AI-based approach aims to democratize antibody discovery against any antigen target of interest . Similarly, microfluidics-enabled screening technologies allow for rapid monoclonal antibody discovery by compartmentalizing single antibody-secreting cells and selecting for specificity . These technologies could potentially:

• Generate more specific DOK3 antibodies with reduced cross-reactivity

• Create a panel of antibodies recognizing different epitopes for comprehensive protein analysis

• Develop phospho-specific antibodies to track DOK3 activation states

• Produce antibodies with improved sensitivity for detecting low-abundance DOK3 in specific cell types

As these technologies mature, researchers can expect higher quality antibody reagents that enable more precise investigation of DOK3 biology and function.

What are common issues when using DOK3 antibody and how can they be addressed?

When working with DOK3 antibody, researchers may encounter several challenges:

• Background signal: Optimize blocking conditions and antibody dilutions; consider using alternative blocking reagents specific to the tissue/cell type

• Weak or absent signal: Verify protein expression in your sample; optimize antigen retrieval methods for IHC-P; adjust antibody concentration

• Non-specific bands in Western blot: Increase antibody dilution; optimize washing steps; consider using gradient gels for better separation

• Variable results between experiments: Standardize protocols, including incubation times and temperatures; prepare fresh working solutions

Proper experimental controls are essential for interpreting results correctly. Include both positive controls (tissues known to express DOK3) and negative controls (primary antibody omission, isotype controls).

How can researchers assess potential cross-reactivity of DOK3 antibody with other DOK family proteins?

DOK family proteins share structural similarities that may lead to antibody cross-reactivity. To assess and mitigate this:

• Sequence comparison: Analyze the immunogen sequence used to generate the antibody against other DOK family members

• Western blot analysis: Test the antibody against recombinant DOK1, DOK2, and other family members

• Knockout validation: When available, use DOK3 knockout samples as negative controls

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity

Similar to approaches used for characterizing anti-idiotypic antibodies in the literature, researchers could develop DOK-specific domain constructs to verify binding specificity of their DOK3 antibody .

How should researchers document and report DOK3 antibody validation in publications?

Proper documentation of antibody validation is essential for research reproducibility. When publishing research using DOK3 antibody, researchers should include:

• Complete antibody information: Manufacturer, catalog number, lot number, RRID when available

• Validation data: Include supplementary data showing specificity tests

• Detailed methods: Provide complete protocols including dilutions, incubation times, buffer compositions

• Control information: Describe all positive and negative controls used

• Batch effects: Note any lot-to-lot variation observed during the study

This level of documentation aligns with emerging standards for antibody reporting in scientific literature and enhances the reproducibility of research findings.

How should quantitative data from DOK3 antibody experiments be analyzed?

Quantitative analysis of DOK3 expression or localization requires rigorous analytical approaches:

• Western blot densitometry: Normalize DOK3 signal to appropriate loading controls; use linear range of detection

• IHC quantification: Consider both staining intensity and percentage of positive cells; use standardized scoring systems

• Immunofluorescence analysis: Apply appropriate background correction; consider co-localization analysis when studying interaction partners

• Statistical considerations: Select appropriate statistical tests based on data distribution; account for technical and biological replicates

For longitudinal studies, consistent analysis methods should be employed across all time points to ensure comparability of results.

How can researchers integrate DOK3 antibody data with other experimental approaches?

DOK3 antibody data is most valuable when integrated with complementary experimental approaches:

• Correlation with functional assays: Link DOK3 expression/localization to cellular function (e.g., B-cell activation, cytokine production)

• Integration with transcriptomic data: Compare protein expression detected by antibody with mRNA expression patterns

• Systems biology approaches: Incorporate DOK3 data into pathway analyses and protein interaction networks

• Temporal analysis: Track changes in DOK3 expression/modification across disease progression or cellular differentiation

This integrative approach provides a more comprehensive understanding of DOK3 biology than antibody-based detection alone.