ASK16 Antibody

What is ASK1 and what cellular pathways does it regulate?

ASK1 (MAP3K5) is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. It plays a critical role in mediating cellular responses to environmental changes and determining cell fate decisions including differentiation and survival. ASK1 is particularly important in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. Additionally, ASK1 is required for innate immune responses, which are essential for host defense against various pathogens. The protein mediates signal transduction of oxidative stress and receptor-mediated inflammatory signals like tumor necrosis factor (TNF) and lipopolysaccharide (LPS). Once activated, ASK1 functions as an upstream activator of both the MKK/JNK and p38 MAPK signal transduction cascades through phosphorylation of several MAP kinase kinases including MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6, and MAP2K7/MKK7 .

What is the significance of phosphorylation at S966 in ASK1 function?

Phosphorylation at serine 966 (S966) represents a key regulatory modification of ASK1. This phosphorylation site is particularly important because it serves as a regulatory switch that can modulate ASK1 activity. When phosphorylated at S966, ASK1 typically shows reduced kinase activity, as this modification can recruit 14-3-3 proteins that sequester ASK1 and prevent its interaction with downstream targets. Researchers studying stress responses, cell death pathways, or inflammatory processes often monitor the phosphorylation status at this site as an indicator of ASK1 regulation. Anti-ASK1 (phospho S966) antibodies specifically target this phosphorylated form, allowing researchers to distinguish between active and inactive states of the protein .

What techniques can ASK1 antibodies be used for in research settings?

Based on available research data, ASK1 antibodies have demonstrated utility in several experimental techniques. The anti-ASK1 (phospho S966) antibody (ab39402) has been validated for immunohistochemistry on paraffin-embedded tissues (IHC-P) with human samples. While the search results don't explicitly mention other techniques, antibodies against signaling proteins like ASK1 are typically employed in Western blotting, immunoprecipitation, flow cytometry, immunofluorescence, and ELISA. The specific applications would depend on the antibody's validation data and the epitope being targeted. Researchers should consult validation studies and literature specific to their ASK1 antibody of interest to determine the full range of applicable techniques .

How should researchers validate ASK1 antibodies prior to experimental use?

Validation of ASK1 antibodies should follow a multi-level approach based on established antibody validation criteria. According to enhanced validation standards, researchers should perform:

• Orthogonal validation - Comparing antibody-based detection with an antibody-independent method like mass spectrometry or RNA expression data to confirm correlation between protein and mRNA levels.

• Independent antibody validation - Using two or more antibodies that target different epitopes of ASK1 to demonstrate consistent staining patterns.

• RNA expression analysis - Comparing antibody staining patterns with RNA expression data to ensure consistency. The RNA similarity score should ideally show high or medium consistency.

• Literature comparison - Ensuring that staining patterns are consistent with published literature on ASK1 expression and localization.

The reliability of antibody data can be classified according to the following criteria:

For rigorous research, antibodies with "Enhanced" or "Supported" reliability scores should be prioritized .

What controls are essential when using phospho-specific ASK1 antibodies?

When working with phospho-specific antibodies such as anti-ASK1 (phospho S966), several critical controls should be implemented:

• Phosphatase treatment control - Treating a portion of your sample with lambda phosphatase to remove phosphorylation, which should eliminate signal from a truly phospho-specific antibody.

• Total ASK1 antibody comparison - Running parallel experiments with antibodies detecting total ASK1 (regardless of phosphorylation state) to normalize phospho-specific signals.

• Positive control samples - Using samples with known ASK1 S966 phosphorylation, such as cells treated with stress inducers that affect this modification.

• Negative control samples - Including samples where the phosphorylation is known to be absent or reduced (e.g., serum-starved cells or cells treated with relevant kinase inhibitors).

• Peptide competition assay - Pre-incubating the antibody with the phosphorylated peptide immunogen to verify signal specificity.

These controls are essential for confirming that signals detected are genuinely from phosphorylated ASK1 at S966 rather than from cross-reactivity with other phosphorylated proteins or non-phosphorylated ASK1 .

How can researchers optimize immunohistochemistry protocols for ASK1 detection?

For optimal ASK1 detection in immunohistochemistry on paraffin-embedded tissues (IHC-P), researchers should consider the following methodological considerations:

• Antigen retrieval optimization - Test different antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0) to determine which best exposes the ASK1 epitope without damaging tissue morphology.

• Antibody titration - Perform a dilution series of the ASK1 antibody to identify the optimal concentration that maximizes specific signal while minimizing background.

• Incubation conditions - Experiment with different incubation times and temperatures (4°C overnight versus room temperature for 1-2 hours) to enhance specific binding.

• Detection system selection - Choose between amplification systems (e.g., biotin-streptavidin, polymer-based) based on the expected abundance of ASK1 in your samples.

• Counterstaining adjustment - Optimize hematoxylin counterstaining to provide clear nuclear definition without obscuring cytoplasmic ASK1 signals.

• Parallel validation - Run a known positive control tissue alongside experimental samples to confirm proper protocol functioning.

Researchers should also be aware that phospho-specific antibodies like anti-ASK1 (phospho S966) may require special handling to preserve phosphorylation status, including the use of phosphatase inhibitors during sample preparation .

How should researchers interpret conflicting results between different ASK1 antibodies?

When faced with inconsistent results between different ASK1 antibodies, researchers should implement a systematic approach to resolve discrepancies:

• Epitope analysis - Determine the exact epitope recognized by each antibody. Antibodies targeting different regions of ASK1 may yield different results due to epitope accessibility, post-translational modifications, or protein interactions.

• Validation status comparison - Assess the validation level of each antibody according to enhanced validation criteria. Prioritize results from antibodies with "Enhanced" reliability scores over those with "Supported" or "Approved" status.

• Application-specific optimization - Consider whether each antibody has been specifically validated for your application. An antibody that works well for Western blotting may not be suitable for immunohistochemistry.

• Independent verification - Employ an orthogonal method that doesn't rely on antibodies (e.g., mass spectrometry or RNA analysis) to determine which antibody results more accurately reflect true ASK1 expression or phosphorylation status.

• Biological context examination - Evaluate whether discrepancies might reflect actual biological differences rather than technical issues. For example, certain stimuli might induce conformational changes that expose or mask specific epitopes.

This approach aligns with the enhanced validation strategy described in the literature, which emphasizes using orthogonal methods and independent antibodies to validate protein expression patterns .

What are common sources of false positives and negatives when using ASK1 phospho-specific antibodies?

Common sources of false results when using phospho-specific antibodies such as anti-ASK1 (phospho S966) include:

False Positives:

• Cross-reactivity with similar phosphorylation motifs on other proteins

• Inadequate blocking leading to non-specific binding

• Post-collection phosphorylation due to delayed fixation or improper sample handling

• Inappropriate antigen retrieval causing exposure of non-specific epitopes

• Secondary antibody cross-reactivity

False Negatives:

• Epitope masking due to protein-protein interactions

• Dephosphorylation during sample processing (insufficient phosphatase inhibitors)

• Overfixation leading to epitope damage or masking

• Inadequate antigen retrieval

• Antibody denaturation or degradation

To minimize these issues, researchers should implement appropriate controls, optimize sample handling to preserve phosphorylation status, and follow validation guidelines such as those described for enhanced antibody validation. This includes performing orthogonal validation and using independent antibodies targeting different epitopes .

How can researchers quantitatively analyze ASK1 phosphorylation in complex samples?

Quantitative analysis of ASK1 phosphorylation requires rigorous methodology:

This quantitative approach enables meaningful comparisons of ASK1 phosphorylation states across different experimental conditions .

How can ASK1 antibodies be utilized to study stress response pathways in disease models?

ASK1 antibodies, particularly phospho-specific variants, serve as powerful tools for investigating stress response pathways in disease models:

• Oxidative stress research - Since ASK1 is activated by reactive oxygen species (ROS), researchers can use phospho-ASK1 antibodies to monitor oxidative stress responses in models of neurodegenerative diseases, cardiovascular disorders, and inflammatory conditions. Decreased phosphorylation at S966 would indicate ASK1 activation in response to oxidative stress.

• Inflammatory pathway analysis - ASK1 mediates responses to inflammatory signals like TNF and LPS. Researchers can use ASK1 antibodies to investigate the role of this kinase in inflammatory diseases by tracking its activation and downstream signaling.

• Apoptosis mechanism elucidation - As ASK1 plays a crucial role in the apoptosis signal transduction pathway, researchers can use antibodies to study the kinetics of ASK1 activation during programmed cell death in cancer models or neurodegenerative diseases.

• Innate immunity studies - Given ASK1's requirement for innate immune responses, researchers can apply ASK1 antibodies to investigate host defense mechanisms against pathogens, potentially identifying novel therapeutic targets.

• Drug development research - Phospho-specific ASK1 antibodies can help evaluate the efficacy of kinase inhibitors or other compounds targeting the ASK1 pathway by monitoring changes in phosphorylation status.

These applications leverage ASK1's central role in stress responses and cell fate determination, making ASK1 antibodies valuable tools for understanding disease mechanisms .

What emerging technologies are enhancing the specificity and sensitivity of ASK1 detection?

Several cutting-edge technologies are improving ASK1 detection capabilities:

• Proximity ligation assays (PLA) - This technique allows visualization of protein-protein interactions involving ASK1 in situ by generating fluorescent signals only when two antibodies targeting different proteins are in close proximity. This approach can reveal ASK1 interactions with regulators like thioredoxin or downstream targets.

• Single-cell Western blotting - This emerging technology enables analysis of ASK1 expression and phosphorylation status at the single-cell level, revealing heterogeneity within populations that might be masked in conventional Western blots.

• Mass cytometry (CyTOF) - By conjugating ASK1 antibodies to metal isotopes rather than fluorophores, researchers can simultaneously detect numerous proteins and phosphorylation sites in single cells without spectral overlap limitations.

• Biophysics-informed modeling for antibody design - Advanced computational approaches are being developed to design antibodies with custom specificity profiles. These methods optimize energy functions to create either cross-specific antibodies (interacting with several distinct ligands) or highly specific antibodies (interacting with a single ligand while excluding others), enabling precise targeting of ASK1 or specific phosphorylated forms .

• Nanobody technology - Single-domain antibodies derived from camelids are being developed against various targets including kinases. Their small size enables access to epitopes that conventional antibodies cannot reach, potentially providing new tools for studying ASK1 conformational states.

These technological advances offer researchers unprecedented opportunities to study ASK1 signaling with improved spatial, temporal, and molecular resolution .

How can ASK1 antibodies be integrated into multi-parameter analyses of signaling networks?

Integration of ASK1 antibodies into multi-parameter signaling analyses requires sophisticated experimental design:

• Multiplexed immunoassays - Researchers can simultaneously measure ASK1 phosphorylation alongside other components of the MAPK pathway (e.g., p38, JNK) and related pathways using multiplexed bead-based assays or microarray platforms. This approach reveals coordination between different signaling branches.

• Sequential immunoblotting - By performing sequential probing of the same membrane with different antibodies (stripping and reprobing), researchers can analyze multiple phosphorylation sites on ASK1 (not just S966) and correlate these with downstream effectors.

• Imaging mass cytometry - This technique combines immunohistochemistry with mass spectrometry to enable simultaneous visualization of dozens of proteins and their modifications in tissue sections, providing spatial context to ASK1 signaling.

• Pathway reconstruction analysis - By collecting quantitative data on ASK1 and related proteins across multiple time points and conditions, researchers can perform computational pathway reconstruction to infer causal relationships and feedback mechanisms.

• Single-cell multi-omics integration - Combining single-cell analysis of ASK1 (using antibodies) with transcriptomics or proteomics data enables correlation between ASK1 activity and global cellular responses at unprecedented resolution.

Such integrative approaches are essential for understanding ASK1's position within the complex web of cellular signaling and for identifying potential points of therapeutic intervention in conditions where ASK1 signaling is dysregulated .

What reproducibility metrics should researchers track when using ASK1 antibodies?

To ensure robust and reliable results with ASK1 antibodies, researchers should monitor several key reproducibility metrics:

• Coefficient of Variation (CV) - For quantitative assays using ASK1 antibodies, calculate and report the CV across technical and biological replicates. Based on established benchmarks for antibody-based assays, researchers should aim for CVs below 15%. For reference, immunoassays for other proteins have achieved CVs of 9.8-14.4% when properly optimized .

• Signal-to-noise ratio - Quantify the ratio between specific ASK1 signal and background. Higher ratios indicate better antibody performance and more reliable detection.

• Lot-to-lot consistency - When obtaining new lots of the same ASK1 antibody, perform comparative analyses to ensure consistent results. Document lot numbers and any observed variations.

• Assay sensitivity and detection limits - Determine the lower and upper limits of detection for your specific application, which helps ensure measurements are taken within the reliable range of the assay.

• Antibody specificity measures - Regularly perform controls to confirm antibody specificity, such as peptide competition assays or testing in systems with ASK1 knockdown/knockout.

Maintaining detailed records of these metrics facilitates troubleshooting and enables more meaningful comparisons across experiments and between laboratories .

How should researchers address unexpected patterns of ASK1 expression or phosphorylation?

When encountering unexpected ASK1 expression or phosphorylation patterns, researchers should follow this systematic troubleshooting approach:

• Technical validation - First rule out technical issues by repeating the experiment with appropriate controls. For phospho-specific detection, include phosphatase-treated controls and positive controls (e.g., cells treated with known ASK1 activators).

• Antibody re-validation - Confirm antibody specificity using orthogonal methods as described in enhanced validation protocols. Consider testing an independent antibody targeting a different epitope to verify the unexpected pattern.

• Biological context assessment - Evaluate whether the unexpected results might represent genuine biological phenomena:

  • Could post-translational modifications be affecting epitope accessibility?

  • Are there tissue-specific or cell type-specific ASK1 isoforms or regulatory mechanisms?

  • Could experimental conditions be inducing feedback loops or compensation mechanisms?

• Literature comparison - Conduct a thorough literature review to determine if similar unexpected patterns have been reported by others, perhaps in different contexts.

• Advanced analysis - If the unexpected pattern persists after validation, employ advanced techniques (e.g., mass spectrometry, RNA-seq) to gain deeper insights into the underlying biology.

This structured approach helps distinguish between technical artifacts and potentially novel biological insights regarding ASK1 regulation or function .

What cross-reactivity issues should researchers be aware of when using ASK1 antibodies?

Researchers should be vigilant about several potential cross-reactivity issues with ASK1 antibodies:

• Homologous protein cross-reactivity - ASK1 (MAP3K5) belongs to a family of related kinases, including ASK2 (MAP3K6) and ASK3 (MAP3K15), which share significant sequence homology. Antibodies targeting conserved domains might cross-react with these related proteins.

• Phosphorylation motif similarity - Phospho-specific antibodies like anti-ASK1 (phospho S966) recognize not just the amino acid sequence but also the phosphorylation. Other proteins with similar phosphorylation motifs might be recognized, especially if the epitope is short.

• Isoform specificity - ASK1 has multiple isoforms resulting from alternative splicing. Researchers should verify whether their antibody recognizes all isoforms or is specific to certain variants.

• Species cross-reactivity - While the antibody mentioned in the search results is validated for human samples, cross-reactivity with ASK1 from other species may vary based on sequence conservation. Researchers working with animal models should verify species reactivity.

• Non-specific binding in certain tissues - Some tissues may present particular cross-reactivity challenges due to high autofluorescence, endogenous peroxidase activity, or abundant similar epitopes.

To address these concerns, researchers should perform thorough validation including Western blots to confirm the antibody detects a protein of the expected molecular weight, peptide competition assays, and testing in systems where ASK1 expression is manipulated (overexpression, knockdown, or knockout) .

How are ASK1 antibodies contributing to our understanding of neurodegenerative diseases?

ASK1 antibodies are providing valuable insights into neurodegenerative pathologies:

• Oxidative stress mechanisms - Using phospho-specific ASK1 antibodies, researchers can track ASK1 activation in response to oxidative stress in models of Alzheimer's, Parkinson's, and Huntington's diseases. This helps establish temporal relationships between oxidative damage, ASK1 activation, and neuronal death.

• Protein aggregate responses - ASK1 antibodies enable the investigation of how protein aggregates (e.g., Aβ, α-synuclein) trigger stress signaling cascades through ASK1 activation, offering potential therapeutic targets for intervention.

• Glial cell involvement - By examining ASK1 expression and activation in different cell types using immunohistochemistry with ASK1 antibodies, researchers can determine whether ASK1 signaling in glial cells contributes to neuroinflammation and subsequent neurodegeneration.

• Drug target validation - ASK1 inhibitors are being investigated as potential treatments for neurodegenerative diseases. Antibodies provide critical tools for validating target engagement and efficacy in preclinical models.

• Biomarker development - Quantitative analysis of phosphorylated ASK1 in accessible fluids or tissues using sensitive immunoassays could potentially serve as biomarkers for disease progression or treatment response.

These applications of ASK1 antibodies are advancing our understanding of the complex molecular mechanisms underlying neurodegenerative processes and may lead to novel therapeutic strategies targeting the ASK1 pathway .

What role do ASK1 antibodies play in immunotherapy research?

ASK1 antibodies serve multiple functions in immunotherapy research:

• Immune cell activation monitoring - ASK1 plays a role in immune cell activation and inflammatory responses. Researchers use ASK1 antibodies to track signaling events in various immune cell populations following stimulation, helping to characterize the molecular basis of immune responses.

• Checkpoint inhibitor mechanism studies - ASK1 antibodies help investigate whether and how immune checkpoint inhibitors affect stress response pathways in immune cells and cancer cells, potentially revealing mechanisms of resistance or adverse effects.

• CAR-T cell optimization - By monitoring ASK1 activation in engineered T cells, researchers can investigate stress responses that might limit CAR-T cell persistence or function, potentially leading to improved cell therapy designs.

• Tumor microenvironment characterization - Multiplexed immunohistochemistry incorporating ASK1 antibodies enables detailed analysis of stress signaling states within the heterogeneous tumor microenvironment, revealing potential targets for combination therapy.

• Therapeutic antibody development methodology - The approaches used to develop and validate specific antibodies against ASK1 inform broader antibody engineering strategies. For example, enhanced validation techniques and biophysics-informed modeling for custom specificity profiles represent methodologies applicable to therapeutic antibody development .

While ASK1 itself may not be a direct target of current immunotherapies, antibodies against ASK1 provide valuable research tools for understanding the complex interplay between stress signaling and immune function .

How can researchers leverage ASK1 antibodies to study the relationship between cellular stress and inflammation?

ASK1 antibodies offer powerful approaches to investigate the stress-inflammation axis:

• Temporal signaling dynamics - Using phospho-specific ASK1 antibodies in time-course experiments, researchers can establish the precise sequence of events linking cellular stressors (e.g., ROS, ER stress) to inflammatory responses. This temporal resolution helps identify the initiating events in stress-induced inflammation.

• Cell type-specific responses - Through immunohistochemistry or flow cytometry with ASK1 antibodies, researchers can map which cell types within complex tissues exhibit ASK1 activation during inflammatory conditions, revealing the cellular architecture of stress responses.

• Feedback loop analysis - ASK1 activation can both respond to and trigger inflammatory mediator production. By simultaneously monitoring ASK1 phosphorylation and inflammatory cytokine levels, researchers can characterize these feedback relationships.

• Subcellular localization studies - Using immunofluorescence with ASK1 antibodies, researchers can track stress-induced changes in ASK1 localization, revealing how compartmentalization regulates inflammatory signaling.

• Intervention point identification - By precisely mapping when and where ASK1 becomes activated during the progression from cellular stress to inflammation, researchers can identify optimal timing for therapeutic interventions targeting this pathway.

This research area is particularly relevant to chronic inflammatory diseases, where persistent cellular stress may drive pathological inflammation. ASK1 sits at a critical junction between stress sensing and inflammatory signal propagation, making antibodies against this kinase invaluable for mechanistic studies .