DOF4.1 Antibody

What is DOK4 and what cellular pathways does it influence?

DOK4 (Downstream of kinase 4, also known as IRS5) is a 41 kDa member of the type B subfamily of the DOK family of proteins. It functions primarily as an intermediary between the insulin receptor (InsR) and downstream MAP kinase activation. DOK4 is expressed in T cells and endothelium, as well as in embryonic neurons and renal anlage during development . The protein contains an N-terminal insulin receptor substrate (IRS) pleckstrin homology domain (spanning amino acids 7-109) and a centrally placed IRS phosphotyrosine-binding domain (amino acids 125-236). This structure enables DOK4 to participate in signaling cascades downstream of receptor tyrosine kinases, making it an important molecule in cellular signal transduction research .

What detection methods work most effectively with DOK4 antibody?

DOK4 antibody has demonstrated effectiveness in multiple detection methods, with Western blotting and immunohistochemistry (IHC) showing particularly robust results. For Western blot applications, DOK4 antibody detects a specific band at approximately 40 kDa under reducing conditions when using appropriate immunoblot buffer systems (e.g., Immunoblot Buffer Group 8) . For IHC applications, optimal results have been achieved using immersion-fixed paraffin-embedded tissue sections with overnight incubation at 4°C at a concentration of 15 μg/mL, followed by appropriate secondary antibody detection systems such as HRP-DAB staining kits . While direct ELISAs also work for detection, researchers should note that cross-reactivity testing indicates specificity for human DOK4 with minimal (<3%) cross-reactivity to other recombinant proteins.

How should DOK4 antibody be stored to maintain optimal activity?

Proper storage is critical for maintaining DOK4 antibody activity. The recommended protocol involves using a manual defrost freezer and avoiding repeated freeze-thaw cycles which can damage antibody structure. Specific storage conditions include:

• 12 months from date of receipt at -20 to -70°C as originally supplied

• 1 month at 2 to 8°C under sterile conditions after reconstitution

• 6 months at -20 to -70°C under sterile conditions after reconstitution

These guidelines ensure minimal degradation of antibody structure and maintenance of binding specificity throughout experimental timeframes.

What considerations should be made when designing Western blot experiments with DOK4 antibody?

When designing Western blot experiments with DOK4 antibody, several methodological considerations are critical for optimal results. The antibody has been validated for detection of a specific band at approximately 40 kDa under reducing conditions . Researchers should implement the following protocol recommendations:

• Use PVDF membrane for protein transfer rather than nitrocellulose for optimal binding

• Employ appropriate positive controls such as lysates from Jurkat human acute T cell leukemia cell line or HeLa human cervical epithelial carcinoma cell line

• Utilize HRP-conjugated Anti-Sheep IgG secondary antibody (like HAF016) for detection

• Conduct experiments under reducing conditions

• Select appropriate immunoblot buffer systems (Immunoblot Buffer Group 8 is recommended)

Additionally, researchers should consider the potential for post-translational modifications of DOK4 that might affect antibody binding or result in band shifts, especially when examining different tissue or cell types.

How do fixation methods affect DOK4 detection in immunohistochemistry?

Fixation methodology significantly impacts DOK4 detection in immunohistochemical applications. Based on available data, immersion fixation with paraffin embedding has proven effective for DOK4 visualization in human kidney tissue . The recommended protocol involves:

• Immersion fixation followed by paraffin embedding of tissue sections

• Application of DOK4 antibody at 15 μg/mL concentration

• Overnight incubation at 4°C for optimal antibody binding

• Detection using an HRP-DAB Cell & Tissue Staining Kit

• Counterstaining with hematoxylin to provide tissue context

Researchers should be aware that alternative fixation methods (e.g., frozen sections, different fixatives) may require protocol optimization. Cross-comparison studies between different fixation methods are recommended when establishing DOK4 staining in new tissue types or experimental models.

What approaches can minimize cross-reactivity concerns with DOK4 antibody?

While DOK4 antibody shows high specificity for human DOK4, minimizing potential cross-reactivity remains important for research integrity. The antibody shows less than 3% cross-reactivity with recombinant proteins in direct ELISAs . To further minimize cross-reactivity concerns:

• Implement rigorous negative controls (secondary antibody only, isotype controls)

• Include knockout or knockdown validation where possible

• Consider pre-adsorption steps with recombinant proteins in high-sensitivity applications

• Optimize antibody concentration through dilution series (particularly important for new tissue types)

• Validate specificity through multiple detection methods (Western blot and IHC)

Additionally, researchers should be aware of the high sequence homology between human and other mammalian DOK4 proteins (human DOK4 shares 96% and 98% amino acid sequence identity with mouse and canine DOK4, respectively, over amino acids 190-326) . This high conservation increases the potential for cross-species reactivity that may need to be accounted for in experimental design.

What are the considerations for studying DOK4 phosphorylation states?

Since DOK4 functions in signaling pathways downstream of receptor tyrosine kinases, its phosphorylation state is critical to its functional activity. When studying DOK4 phosphorylation:

• Consider using phosphatase inhibitors in lysate preparation to preserve phosphorylation states

• Employ phosphorylation-specific antibodies in conjunction with total DOK4 antibody

• Implement cell stimulation protocols (e.g., insulin or growth factor treatment) to induce phosphorylation changes

• Use appropriate positive controls with known phosphorylation states

• Consider comparing migration patterns on Western blots before and after phosphatase treatment

Researchers should be aware that the binding epitope of the DOK4 antibody (Asp190-Gly326) may be affected by proximal phosphorylation events, potentially altering detection efficiency under different cellular activation states.

How can DOK4 antibody be leveraged in multiplex immunoassays?

Multiplex immunoassays involving DOK4 antibody require careful consideration of antibody compatibility, fluorophore selection, and potential cross-reactivity. Based on experiences with similar antibody systems:

• When selecting secondary antibodies, consider species compatibility issues (sheep anti-human DOK4 antibody requires anti-sheep secondary reagents)

• For fluorescent applications, test for potential spectral overlap between fluorophores

• In co-localization studies, validate antibody specificity with single-staining controls

• Implement appropriate blocking protocols to minimize non-specific binding

• Consider sequential rather than simultaneous staining when using antibodies with similar host species

Notably, while not directly addressing DOK4 antibody, the methodological approaches described for antibody-based assays in search result , including optimization of binding conditions and assessment of binding kinetics through techniques like surface plasmon resonance and biolayer interferometry, provide valuable insights applicable to DOK4 antibody multiplex applications.

What protein preparation techniques optimize DOK4 antibody performance?

Protein preparation is critical for maximizing DOK4 antibody detection sensitivity. Based on available data and general antibody methodologies:

• For cell lysate preparation, use lysis buffers containing appropriate detergents (e.g., RIPA buffer) supplemented with protease inhibitors

• For tissue samples, implement mechanical homogenization followed by detergent-based protein extraction

• Maintain cold chain throughout sample preparation to minimize protein degradation

• Consider subcellular fractionation approaches when studying compartment-specific DOK4 localization

• For Western blot applications, determine optimal protein loading concentration through dilution series

These approaches ensure maximal epitope preservation and accessibility, optimizing DOK4 antibody performance across different experimental platforms.

How do different secondary detection systems impact DOK4 antibody sensitivity?

The choice of secondary detection system significantly impacts DOK4 antibody sensitivity and specificity. Based on validated protocols:

• For Western blot applications, HRP-conjugated anti-sheep IgG secondary antibody has demonstrated effective detection

• For IHC applications, HRP-DAB Cell & Tissue Staining Kit provides robust visualization with appropriate counterstaining

• For fluorescent applications, fluorophore-conjugated secondary antibodies with appropriate spectral properties should be selected

• Amplification systems (e.g., tyramide signal amplification) may be considered for low-abundance DOK4 detection

• Biotin-streptavidin systems offer another alternative with potential sensitivity benefits

Each system presents different sensitivity/background tradeoffs that should be empirically determined for specific experimental conditions.

What troubleshooting approaches address common DOK4 antibody detection issues?

When encountering detection issues with DOK4 antibody, systematic troubleshooting approaches include:

• For weak or absent signal:

  • Increase antibody concentration

  • Extend incubation time

  • Implement epitope retrieval for fixed tissues

  • Verify sample preparation integrity

• For high background:

  • Increase blocking duration and concentration

  • Reduce primary and secondary antibody concentrations

  • Implement additional wash steps

  • Test alternative blocking reagents

• For non-specific bands in Western blot:

  • Optimize sample preparation to reduce protein degradation

  • Adjust gel percentage to better resolve proteins in the 40 kDa range

  • Implement more stringent wash conditions

  • Consider gradient gels for better resolution

These approaches systematically address the most common technical issues encountered in DOK4 antibody applications.

How does DOK4 interact with other signaling proteins in experimental systems?

DOK4 functions as an intermediary between the insulin receptor and downstream MAP kinase activation . When designing experiments to study these interactions:

• Consider co-immunoprecipitation approaches to identify direct binding partners

• Implement proximity ligation assays to visualize protein-protein interactions in situ

• Utilize pathway inhibitors to delineate the signaling cascade involving DOK4

• Design siRNA or CRISPR-based knockdown/knockout experiments to establish functional relationships

• Employ phosphorylation-specific antibodies to track signaling dynamics

These approaches enable comprehensive mapping of DOK4's role in cellular signaling networks and provide mechanistic insights into its function in normal physiology and disease states.

What controls are essential for validating DOK4 antibody specificity?

Rigorous validation of DOK4 antibody specificity requires implementation of multiple controls:

• Positive controls: Jurkat human acute T cell leukemia and HeLa human cervical epithelial carcinoma cell lines have been validated for DOK4 expression

• Negative controls: Cell lines with minimal DOK4 expression, ideally including DOK4 knockout models

• Isotype controls: Non-specific sheep IgG at equivalent concentrations

• Secondary-only controls: Omitting primary antibody to assess non-specific secondary binding

• Peptide competition/pre-adsorption: Pre-incubating antibody with recombinant DOK4 protein to block specific binding

Implementation of these controls strengthens research validity and addresses potential reviewer concerns in publication contexts.

How can researchers optimize DOK4 antibody for flow cytometry applications?

While the search results don't specifically address flow cytometry applications for DOK4 antibody, general principles for adapting antibodies for flow cytometry include:

• Optimize fixation conditions to preserve epitope while maintaining cellular integrity

• Determine appropriate permeabilization protocols for intracellular DOK4 detection

• Establish titration curves to identify optimal antibody concentration

• Implement comprehensive compensation controls when using multiple fluorophores

• Use calibration beads to standardize fluorescence intensity across experiments

Researchers should conduct preliminary validation experiments comparing DOK4 antibody performance in flow cytometry against established detection methods like Western blotting to ensure consistent detection specificity.

What are the consensus best practices for DOK4 antibody-based research?

Based on the available data and general principles of antibody-based research, consensus best practices for DOK4 antibody applications include:

• Rigorous validation through multiple detection methods (Western blot, IHC, ELISA)

• Implementation of appropriate positive and negative controls in each experiment

• Careful attention to storage conditions to maintain antibody integrity

• Optimization of sample preparation protocols specific to each experimental application

• Consistent documentation of antibody lot numbers, dilutions, and incubation conditions

• Consideration of potential cross-reactivity when interpreting results

Adherence to these best practices ensures research reproducibility and facilitates meaningful comparison across different studies involving DOK4.

What emerging technologies might enhance DOK4 antibody applications?

Emerging technologies that may enhance DOK4 antibody applications include:

• Super-resolution microscopy for nanoscale visualization of DOK4 localization

• Mass cytometry (CyTOF) for high-dimensional analysis of DOK4 in cellular subpopulations

• Antibody engineering approaches like those used for penpulimab to enhance stability and reduce non-specific interactions

• Single-cell proteomics for assessing DOK4 expression heterogeneity

• Multiplex spatial proteomics for contextualizing DOK4 expression within tissue architecture