ASK11 Antibody

What is ASK1 and why is it significant in cell signaling research?

ASK1, also known as Mitogen-activated protein kinase kinase kinase 5 (MAP3K5), is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. It plays a pivotal role in the cascades of cellular responses triggered by environmental changes and mediates signaling for cell fate determination, including differentiation and survival. ASK1 is particularly significant in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. Additionally, ASK1 is required for innate immune responses, which are crucial for host defense against various pathogens. The protein mediates signal transduction of various stressors, including oxidative stress and receptor-mediated inflammatory signals like tumor necrosis factor (TNF) or lipopolysaccharide (LPS) . Understanding ASK1 function is critical for researchers studying cellular stress responses, as it activates downstream mitogen-activated protein (MAP) kinase pathways, particularly the JNK and p38 pathways .

What are the structural and functional domains of ASK1 that antibodies typically target?

ASK1 is a 155 kDa protein containing multiple functional domains that antibodies can target. Common epitope regions include:

• C-terminal region (aa 1356-1375 in humans): This region is targeted by many commercially available antibodies, including polyclonal antibodies that recognize the C-terminus .

• N-terminal regulatory domain: Contains binding sites for thioredoxin, which regulates ASK1 activity under oxidative stress conditions.

• Kinase domain: The catalytic region responsible for phosphorylation activity.

• Coiled-coil domains: Mediate protein-protein interactions that regulate ASK1 activity.

Researchers should note that some peptide sequences used as immunogens for antibody development may differ between human and mouse ASK1, particularly in the C-terminal region where the last two amino acids can vary . This variation can affect cross-reactivity of antibodies between species.

How do monoclonal and polyclonal ASK1 antibodies differ in research applications?

What are the optimal conditions for ASK1 antibody usage in Western blotting?

For optimal detection of ASK1 via Western blotting, researchers should consider the following protocol:

• Sample preparation:

  • Use freshly prepared cell lysates when possible

  • Include phosphatase inhibitors if studying ASK1 phosphorylation states

  • Recommended lysis buffer: RIPA buffer supplemented with protease inhibitors

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE to achieve good separation of the 155kDa ASK1 protein

  • Load 20-50μg of total protein per lane

• Transfer conditions:

  • Wet transfer is preferable for large proteins like ASK1

  • Transfer at 30V overnight at 4°C to ensure complete transfer

• Blocking and antibody incubation:

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

  • Primary antibody dilution: 1:1000 for most commercial ASK1 antibodies

  • Incubate with primary antibody overnight at 4°C

  • Secondary antibody dilution: 1:5000, incubate for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) detection systems work well

  • Alternative: Conjugated antibodies like ASK1 Antibody (F-9) HRP can eliminate the need for secondary antibodies

When troubleshooting, note that ASK1 can sometimes appear as multiple bands due to post-translational modifications or proteolytic cleavage. The expected molecular weight is approximately 155kDa, but phosphorylated forms may show slightly altered migration patterns.

How can ASK1 antibodies be effectively used in immunofluorescence and immunohistochemistry?

For successful immunofluorescence (IF) and immunohistochemistry (IHC) experiments with ASK1 antibodies:

Immunofluorescence protocol:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 5 minutes

• Blocking: 1-2% BSA or 5-10% normal serum from the species of secondary antibody

• Primary antibody: Dilute ASK1 antibody 1:100-1:500 in blocking buffer, incubate overnight at 4°C

• Secondary antibody: Use appropriate fluorescently-labeled secondary antibody (1:500-1:1000)

• Counterstain: DAPI for nuclear visualization

• Mounting: Use anti-fade mounting medium

Immunohistochemistry considerations:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is often necessary

• Endogenous peroxidase quenching: 3% hydrogen peroxide for 10 minutes

• Blocking: 5-10% normal serum

• Primary antibody: ASK1 antibody (1:100-1:200), incubate overnight at 4°C

• Detection system: Biotin-streptavidin-HRP or polymer-based detection systems

For both techniques, researchers should note that ASK1 typically shows cytoplasmic localization, but interaction with 14-3-3 proteins can alter its distribution to the perinuclear endoplasmic reticulum region . Control experiments should include omission of primary antibody and ideally tissue from ASK1 knockout models.

What approaches can be used to study ASK1 activation in response to cellular stress?

ASK1 activation can be monitored through several complementary approaches:

• Phosphorylation analysis:

  • ASK1 activation involves auto-phosphorylation at Thr845 (human)

  • Use phospho-specific ASK1 antibodies in Western blotting

  • Compare phospho-ASK1 to total ASK1 levels for quantification

• Downstream signaling detection:

  • Monitor phosphorylation of direct ASK1 substrates (MKK4, MKK3, MKK6)

  • Assess activation of downstream JNK and p38 MAPK pathways

  • Multi-color flow cytometry can be used for single-cell analysis of pathway activation

• Interaction analysis:

  • Co-immunoprecipitation with ASK1 antibodies to detect:

    • Dissociation from inhibitory proteins (Thioredoxin, 14-3-3)

    • Association with activating proteins (TRAF2/6)

  • Proximity ligation assays to visualize protein interactions in situ

• Functional readouts:

  • Apoptosis assays (Annexin V/PI staining, caspase activation)

  • ROS detection using fluorescent probes (DCF-DA)

  • Cell viability assays following oxidative stress induction

• Genetic approaches:

  • CRISPR/Cas9-mediated mutation of key ASK1 regulatory sites

  • Expression of dominant-negative ASK1 mutants

  • RNA interference to reduce ASK1 expression

For studying stress-induced ASK1 activation, common inducers include hydrogen peroxide (0.1-1mM), TNF-α (10-50ng/ml), or UV irradiation. Time-course experiments are crucial, as ASK1 activation can be transient, often peaking between 15-60 minutes after stimulus application.

What are common issues when detecting ASK1 in Western blots and how can they be resolved?

When working with stress-activated proteins like ASK1, it's crucial to control the activation state of your samples. Unstressed control samples should be processed identically but without the activating stimulus. For definitive identification of ASK1-specific bands, using lysates from ASK1 knockout or knockdown cells as negative controls is highly recommended.

How can specificity of ASK1 antibodies be verified in experimental systems?

Verifying antibody specificity is critical for reliable research outcomes. For ASK1 antibodies, researchers should employ multiple validation approaches:

• Genetic validation:

  • Use ASK1 knockout or knockdown cells/tissues as negative controls

  • Overexpression systems with tagged ASK1 as positive controls

  • CRISPR/Cas9-edited cell lines with epitope modifications

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • The specific signal should be significantly reduced or eliminated

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes of ASK1

  • Consistent results across antibodies increase confidence in specificity

• Immunoprecipitation-Western blot approach:

  • Immunoprecipitate with one ASK1 antibody, then detect with another

  • Confirms recognition of the same protein by independent antibodies

• Mass spectrometry validation:

  • Immunoprecipitate ASK1 and analyze by mass spectrometry

  • Confirms identity of the detected protein

For cross-reactivity concerns, particularly between closely related MAP3K family members, specific attention should be paid to the antibody epitope. Antibodies raised against unique regions of ASK1, such as the C-terminal peptide corresponding to aa 1356-1375, offer better specificity than those targeting conserved domains .

What controls should be included when using ASK1 antibodies in immunoprecipitation experiments?

Robust immunoprecipitation (IP) experiments with ASK1 antibodies require thorough controls:

• Essential negative controls:

  • Isotype control: Use matched isotype antibody from same species

  • No-antibody control: Perform IP procedure without adding any antibody

  • Knockout/knockdown control: Use ASK1-deficient samples when available

• Positive controls:

  • Input sample: Load 5-10% of pre-IP lysate to confirm protein presence

  • ASK1-overexpressing cells: Provides strong signal for optimization

  • Known interacting proteins: Co-IP of established partners (e.g., TRAF2, Trx1)

• Validation controls:

  • Reciprocal IP: IP with antibody against known ASK1-interacting protein

  • Sequential IP: Deplete sample with one antibody, then IP with another

  • Transfection controls: Tagged ASK1 constructs for antibody-independent detection

• Technical considerations:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • For monoclonal antibodies like F-9, consider using conjugated agarose forms for direct IP

  • Include protease and phosphatase inhibitors in all buffers

  • Gentle elution conditions to maintain protein interactions

When performing co-IP to study ASK1 interactions, cellular stimulation may be necessary to capture transient interactions. For example, oxidative stress induction (0.5mM H₂O₂ for 15-30 minutes) can enhance interactions with stress-responsive partners.

How can custom specificity profiles be designed for ASK1 antibodies in complex experimental systems?

Designing antibodies with custom specificity profiles for ASK1 is an advanced approach that allows researchers to target specific aspects of ASK1 biology. This process involves:

• Computational modeling approaches:

  • Energy functions can be used to optimize antibody sequences for specific binding profiles

  • For cross-specific sequences (binding to several distinct ligands), jointly minimize the energy functions associated with desired ligands

  • For highly specific sequences, minimize energy functions for desired ligands while maximizing those for undesired ligands

• Phage display selection strategies:

  • Use phage display experiments with negative selection against closely related proteins

  • Implement alternating positive and negative selection rounds to improve specificity

  • Consider competitive elution with specific ASK1 domains or peptides

• Epitope mapping and targeting:

  • Target unique regions of ASK1 that differ from related MAP3K family members

  • Focus on regions that undergo conformational changes upon activation

  • Consider post-translational modification sites specific to particular ASK1 states

• Validation in complex samples:

  • Test antibody performance in mixtures containing potential cross-reactive proteins

  • Evaluate specificity across different cell types and tissues

  • Assess performance under different experimental conditions (native vs. denatured)

For researchers developing custom antibodies, it's essential to track the performance of each antibody variant systematically. Creating a standardized ontology for multiformat antibody design helps manage various components and track how design alterations impact efficacy and specificity .

What methodologies can detect different activation states of ASK1 in pathological conditions?

Detecting specific ASK1 activation states in pathological contexts requires sophisticated approaches:

• Phosphorylation-state specific antibodies:

  • Target key regulatory phosphorylation sites (e.g., Thr845 for activation, Ser83 for inhibition)

  • Validate antibody specificity with phosphatase treatments and phosphomimetic mutants

  • Use for Western blot, immunohistochemistry, and flow cytometry applications

• Conformation-sensitive antibodies:

  • Develop antibodies that specifically recognize activated ASK1 conformations

  • Screen for antibodies that preferentially bind to stress-induced ASK1 states

  • Validate with purified recombinant ASK1 in different conformational states

• Proximity-based detection methods:

  • Proximity ligation assays (PLA) to visualize ASK1 interactions in situ

  • FRET-based biosensors to monitor ASK1 activation in live cells

  • BiFC (Bimolecular Fluorescence Complementation) to detect specific protein complexes

• Multi-parameter analysis:

  • Combine ASK1 activation markers with disease-specific markers

  • Use multiplexed immunofluorescence or mass cytometry (CyTOF)

  • Perform single-cell analysis to identify cell populations with activated ASK1

• Activity-based probes:

  • Develop covalent inhibitors modified for visualization

  • Use activity-based protein profiling approaches

  • Combine with mass spectrometry for proteome-wide analysis

These methods can be particularly valuable for studying ASK1's role in diseases like neurodegenerative disorders and cancer, where dysregulation of ASK1 signaling has been implicated . When applying these techniques to patient samples, appropriate controls must include both healthy tissues and disease-relevant models where ASK1 signaling has been genetically or pharmacologically modulated.

How can ASK1 antibodies be used to investigate protein-protein interactions in the MAP kinase cascade?

Investigating ASK1's interactions within the MAP kinase cascade requires specialized applications of antibodies:

• Co-immunoprecipitation strategies:

  • Use ASK1 antibodies conjugated to solid supports like agarose

  • Sequential co-IP: IP ASK1 first, then detect associated proteins

  • Crosslinking co-IP for transient interactions: Use cell-permeable crosslinkers before lysis

  • IP under native conditions to preserve protein complexes

• Proximity-based interaction methods:

  • Proximity ligation assay (PLA): Visualize interactions between ASK1 and partners in situ

  • FRET/BRET approaches: Detect direct protein interactions in living cells

  • BioID or APEX2 proximity labeling: Identify proteins in the vicinity of ASK1

• Domain-specific interaction analysis:

  • Map binding domains using truncated proteins and domain-specific antibodies

  • Peptide arrays probed with purified proteins and detected with antibodies

  • Competition assays with domain-specific peptides

• Functional validation of interactions:

  • Mutate key residues in interaction interfaces

  • Use domain-specific blocking antibodies to disrupt specific interactions

  • Employ cell-permeable peptides mimicking interaction domains

• Quantitative interaction analysis:

  • IP-mass spectrometry for unbiased interaction profiling

  • Surface plasmon resonance with purified components

  • AlphaLISA or HTRF for high-throughput screening of modulators

When studying ASK1 interactions with downstream components like MEK-4 and MEK-3 , it's important to consider the activation state of the pathway. Using appropriate stimuli (oxidative stress, cytokines) can enhance detection of stimulus-dependent interactions. For example, after TNF-α treatment, ASK1 association with TRAF2 increases, while thioredoxin dissociates under oxidative stress.

How are ASK1 antibodies being used to study its role in neurodegenerative diseases?

ASK1 has emerged as a significant target in neurodegenerative disease research, with antibodies playing crucial roles in elucidating its pathological involvement:

• Cellular localization studies:

  • Immunohistochemical analysis of ASK1 in patient brain tissues

  • Co-localization with disease-specific protein aggregates (Aβ, tau, α-synuclein)

  • Comparison of subcellular distribution between healthy and diseased neurons

• Activation status assessment:

  • Monitoring phospho-ASK1 (Thr845) levels in disease models and patient samples

  • Correlation of ASK1 activation with disease progression markers

  • Single-cell analysis of ASK1 activation in specific neuronal populations

• Mechanistic studies:

  • Investigation of ASK1-mediated neuronal death pathways

  • Analysis of interactions between ASK1 and disease-specific proteins

  • Examination of the relationship between oxidative stress, ASK1 activation, and neurodegeneration

• Therapeutic target validation:

  • Antibody-mediated inhibition of ASK1 in cellular and animal models

  • Pharmacodynamic biomarker development using phospho-specific antibodies

  • Evaluation of ASK1 inhibitors' effects on downstream signaling

• Biomarker development:

  • Exploration of phospho-ASK1 as a potential biomarker in CSF or blood

  • Correlation of ASK1 activation with clinical outcomes

  • Development of sensitive detection methods for ASK1 pathway activation

Researchers investigating ASK1 in Alzheimer's, Parkinson's, or ALS should consider using multiple antibodies targeting different epitopes and activation states to build a comprehensive picture of ASK1's role in disease pathogenesis . Time-course experiments are particularly valuable, as ASK1 activation may precede overt pathology and clinical symptoms.

What methodological approaches can detect ASK1-mediated cellular responses in immune cell populations?

Investigating ASK1 function in immune cells requires specialized approaches:

• Flow cytometry-based methods:

  • Intracellular staining for phospho-ASK1 and downstream targets

  • Multi-parameter analysis combining ASK1 activation with immune cell markers

  • Phospho-flow cytometry for single-cell analysis of signaling dynamics

• Imaging techniques:

  • Imaging flow cytometry to visualize ASK1 localization in immune cell subsets

  • Multiplexed immunofluorescence to detect ASK1 activation in tissue-resident immune cells

  • Live-cell imaging with fluorescent reporters to track signaling in real-time

• Functional readouts:

  • Correlation of ASK1 activation with cytokine production

  • Assessment of immune cell differentiation following ASK1 manipulation

  • Analysis of ASK1's impact on immune cell migration and effector functions

• Single-cell approaches:

  • Single-cell RNA-seq combined with protein analysis (CITE-seq)

  • Single-cell western blotting for ASK1 pathway components

  • Mass cytometry (CyTOF) for high-dimensional analysis of signaling networks

• In vivo analysis:

  • Adoptive transfer experiments with ASK1-deficient immune cells

  • In vivo imaging of ASK1 reporter mice during immune challenges

  • Investigation of ASK1 activation in immune cells during disease pathogenesis

ASK1 plays critical roles in innate immune responses, which are essential for host defense against various pathogens . When studying age-associated B cells (ABCs), which are linked to autoimmunity and aging, researchers should monitor ASK1 activation in the context of B cell receptor signaling and TLR stimulation . This is particularly important when investigating how ABCs differentiate into antibody-secreting cells upon antigen rechallenge.

How can researchers design experiments to resolve contradictory findings about ASK1 function in different cell types?

Resolving contradictions in ASK1 research requires systematic experimental approaches:

• Standardized experimental systems:

  • Use identical experimental conditions across cell types

  • Implement consistent activation protocols and timepoints

  • Employ the same antibodies and detection methods

• Comprehensive cell type analysis:

  • Compare primary cells, cell lines, and in vivo models

  • Analyze multiple cell types simultaneously under identical conditions

  • Consider tissue-specific isoforms or splice variants of ASK1

• Context-dependent signaling analysis:

  • Investigate ASK1 signaling network differences between cell types

  • Map cell-type-specific ASK1 interactome using IP-mass spectrometry

  • Examine expression levels of upstream regulators and downstream effectors

• Genetic approaches:

  • Generate conditional ASK1 knockout models for tissue-specific deletion

  • Use CRISPR/Cas9 to introduce identical mutations across cell types

  • Employ rescue experiments with wild-type and mutant ASK1

• Quantitative analysis:

  • Develop quantitative models of ASK1 signaling dynamics

  • Measure absolute protein concentrations across cell types

  • Perform dose-response and time-course analyses

• Multi-omics integration:

  • Combine phosphoproteomics, transcriptomics, and metabolomics

  • Identify cell-type-specific signaling networks

  • Use systems biology approaches to model differential responses

When encountering contradictory results, researchers should systematically test hypotheses about why ASK1 might function differently across contexts. For example, the balance between pro-survival and pro-apoptotic ASK1 signaling may depend on expression levels of specific regulatory proteins, metabolic state, or interaction with other signaling pathways. Carefully designed experiments with appropriate controls and quantitative readouts are essential for resolving these contradictions.