AXS1 Antibody

What is ASK1 and why is it important in cancer research?

ASK1 (Apoptosis Signal-regulating Kinase 1) is a serine/threonine kinase that plays critical roles in cellular stress responses and apoptotic signaling pathways. Its phosphorylation status, particularly at sites like Ser966, regulates its activity and downstream effects. In cancer research, ASK1 signaling has been implicated in both tumor suppression and progression, depending on the cancer type and cellular context. For instance, ASK1 can mediate tumor suppression through stress-induced apoptosis in certain cancers . Understanding ASK1's role requires specific antibodies that can detect both total ASK1 protein and its various phosphorylated forms to elucidate its activation state in experimental models.

What types of ASK1 antibodies are available for research applications?

Multiple types of ASK1 antibodies are available for research applications, including:

• Antibodies detecting total ASK1 protein regardless of phosphorylation status

• Phospho-specific antibodies that detect ASK1 only when phosphorylated at specific residues, such as:

  • Anti-ASK1 (phospho-S966) antibodies

  • Anti-ASK1 (phospho-Ser83) antibodies

  • Anti-ASK1 (phospho-Ser1033) antibodies

  • Anti-ASK1 (phospho-Thr918) antibodies

  • Anti-ASK1 (phospho-Ser967) antibodies
    These antibodies are typically available in different host species (commonly rabbit) and may be monoclonal or polyclonal depending on the supplier and specific application requirements .

What are the common applications for ASK1 antibodies in research?

ASK1 antibodies are commonly used in several research applications:

• Western blotting (WB): For detecting ASK1 protein expression levels and phosphorylation status in cell or tissue lysates

• Immunohistochemistry (IHC): For visualizing ASK1 expression and localization in tissue sections

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection of ASK1 protein levels

• Immunoprecipitation (IP): For isolating ASK1 protein complexes to study protein-protein interactions

• Protein signaling studies: For investigating ASK1 activation in response to various cellular stresses
  The choice of application dictates which specific ASK1 antibody is most appropriate for a given experimental setup.

How do I determine the appropriate ASK1 antibody dilution for Western blotting?

Determining the appropriate dilution for ASK1 antibodies in Western blotting requires careful optimization:

• Start with the manufacturer's recommended dilution range (typically 1:500 to 1:2000 for primary antibodies)

• Perform a dilution series experiment using positive control samples known to express ASK1

• Include negative controls (samples without ASK1 expression) to assess background signal

• Evaluate signal-to-noise ratio at each dilution

• Consider that phospho-specific ASK1 antibodies may require different dilutions than total ASK1 antibodies

• Document the optimal dilution in your laboratory protocols for reproducibility
  For ASK1 phospho-specific antibodies (like anti-phospho-S966), additional optimization may be necessary depending on the phosphorylation state of your samples and the specificity of the antibody .

How can I validate the specificity of phospho-ASK1 antibodies in my experimental system?

Validating phospho-ASK1 antibody specificity is critical for accurate interpretation of results:

• Phosphatase treatment controls: Treat a portion of your lysate with lambda phosphatase to remove phosphorylation and confirm loss of signal with phospho-specific antibodies

• Stimulation/inhibition experiments: Use known activators or inhibitors of ASK1 phosphorylation to demonstrate appropriate signal changes

• siRNA or CRISPR knockdown: Reduce ASK1 expression and confirm corresponding reduction in antibody signal

• Peptide competition assays: Pre-incubate the antibody with the immunizing phosphopeptide to block specific binding

• Multiple antibody comparison: Use antibodies from different sources or clones that recognize the same phospho-site

• Mass spectrometry validation: For critical experiments, confirm phosphorylation status using mass spectrometry
  Phospho-specific ASK1 antibodies, such as those detecting Ser966 phosphorylation, require particular attention to validation as their specificity directly impacts data interpretation .

What are the best experimental approaches to study ASK1 phosphorylation dynamics in cancer models?

To effectively study ASK1 phosphorylation dynamics in cancer:

• Time-course experiments: Analyze phosphorylation changes following treatment with relevant stimuli

• Multi-site phosphorylation analysis: Use different phospho-specific antibodies (e.g., phospho-S966, phospho-S83) to monitor different regulatory sites simultaneously

• In vitro kinase assays: Assess ASK1 activity using purified protein and substrates

• Proximity ligation assays: Visualize ASK1 interactions with regulatory partners in situ

• Live-cell imaging: Use fluorescent reporters coupled to phospho-binding domains to track ASK1 activation in real-time

• Correlation with downstream signaling: Monitor ASK1 phosphorylation alongside activation of downstream targets

• Comparison across cell lines: Analyze ASK1 phosphorylation patterns in cancer cell lines with different characteristics
  These approaches can reveal how ASK1 regulation differs between normal and cancer cells, potentially identifying therapeutic opportunities .

How does ASK1 phosphorylation at different sites affect its function in tumor progression?

ASK1 phosphorylation at different sites has distinct functional consequences:

• Ser966/Ser967 phosphorylation: Often associated with inhibition of ASK1 activity through 14-3-3 protein binding

• Thr838 phosphorylation: Associated with ASK1 activation and promotion of apoptosis

• Ser83 phosphorylation: Can be mediated by Akt and generally inhibits ASK1 activity

• Site-specific effects on cancer progression:

  • In some cancer models, inhibitory phosphorylation (e.g., at Ser966) may promote survival and resistance to apoptosis

  • Reduced activating phosphorylation may prevent ASK1-mediated cell death
    Understanding these site-specific modifications is crucial when designing experiments to modulate ASK1 activity in cancer research .

How can I differentiate between ASK1 and annexin-A1 (ANXA1) antibody targets in my research?

Despite similar abbreviations, ASK1 and ANXA1 are distinct proteins requiring careful experimental design:

• Target verification:

  • ASK1 (Apoptosis Signal-regulating Kinase 1) is a ~155 kDa serine/threonine kinase

  • ANXA1 (Annexin-A1) is a ~37 kDa immunomodulatory protein

• Differential detection strategies:

  • Use antibodies with confirmed specificity for each target

  • Verify by molecular weight on Western blots (ASK1: ~155 kDa; ANXA1: ~37 kDa)

  • Perform immunoprecipitation with target-specific antibodies followed by mass spectrometry validation

• Functional validation:

  • ASK1 inhibition affects stress signaling pathways

  • ANXA1 targeting (e.g., with MDX-124 antibody) affects cell cycle progression at G1 phase
    This differentiation is particularly important when studying cancer models where both proteins may play significant roles .

What are the optimal sample preparation methods for detecting phosphorylated ASK1?

Optimal sample preparation for phosphorylated ASK1 detection requires:

• Rapid sample processing to prevent phosphatase activity:

  • Flash freeze tissues immediately after collection

  • Process cell lysates on ice with pre-chilled buffers

• Phosphatase inhibitor inclusion:

  • Use fresh phosphatase inhibitor cocktails containing sodium fluoride, sodium orthovanadate, and β-glycerophosphate

  • Consider addition of calyculin A or okadaic acid for serine/threonine phosphatases

• Lysis buffer optimization:

  • Use RIPA or modified RIPA buffers with sufficient ionic strength

  • Include 1% NP-40 or Triton X-100 for membrane protein solubilization

  • Maintain pH between 7.2-7.4

• Standardized lysate handling:

  • Avoid multiple freeze-thaw cycles

  • Determine protein concentration before immunoblotting

  • Load equal amounts of protein (typically 20-50 μg) per lane
    These precautions preserve phosphorylation status for accurate assessment of ASK1 activation state.

How do I troubleshoot weak or absent signals when using ASK1 phospho-antibodies?

When troubleshooting weak or absent signals with ASK1 phospho-antibodies:

• Sample-related issues:

  • Confirm ASK1 expression in your model system

  • Verify phosphorylation status with positive controls

  • Ensure phosphatase inhibitors were effective during sample preparation

• Antibody-related solutions:

  • Titrate antibody concentration (try higher concentrations)

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

  • Test different blocking agents (BSA vs. milk proteins)

  • Consider alternative antibody clones or vendors

• Detection system optimization:

  • Use high-sensitivity ECL substrates for chemiluminescence

  • Increase exposure time for imaging

  • Try signal amplification methods (e.g., biotin-streptavidin systems)

• Protocol modifications:

  • Optimize transfer conditions for high molecular weight proteins

  • Consider using PVDF rather than nitrocellulose membranes

  • Test different antigen retrieval methods for IHC applications
    Documentation of optimization steps ensures reproducibility across experiments.

What controls should be included when using ASK1 phospho-specific antibodies?

Essential controls for experiments using ASK1 phospho-specific antibodies include:

• Positive controls:

  • Lysates from cells treated with known ASK1 activators (e.g., H₂O₂, TNF-α)

  • Recombinant phosphorylated ASK1 protein (if available)

• Negative controls:

  • Lysates from ASK1 knockdown or knockout cells

  • Phosphatase-treated samples to remove phosphorylation

  • Isotype control antibodies to assess non-specific binding

• Loading controls:

  • Total protein stains (e.g., Ponceau S, REVERT)

  • Housekeeping proteins (e.g., β-actin, GAPDH)

• Technical controls:

  • Include both phospho-specific and total ASK1 antibodies

  • Use the same samples on parallel blots or after stripping and reprobing

  • Include molecular weight markers to confirm target size
    These controls allow proper interpretation of results and troubleshooting of technical issues.

How can ASK1 antibodies be effectively used in co-immunoprecipitation studies?

For effective co-immunoprecipitation (co-IP) studies with ASK1 antibodies:

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation

  • Consider using different antibodies for IP and detection (different epitopes)

  • For phospho-studies, determine whether phospho-specific antibodies maintain binding during IP conditions

• Lysis conditions:

  • Use gentler lysis buffers (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions

  • Optimize salt concentration (typically 100-150 mM NaCl)

  • Include protease and phosphatase inhibitors

• IP procedure:

  • Pre-clear lysates with protein A/G beads

  • Use sufficient antibody (2-5 μg per mg of protein lysate)

  • Optimize incubation time (4-16 hours at 4°C)

  • Include appropriate controls (IgG control, input samples)

• Detection strategy:

  • Blot for interaction partners using specific antibodies

  • Consider using IP kits designed for co-IP applications

  • For weak interactions, consider crosslinking approaches
    This methodology can reveal ASK1 binding partners and how phosphorylation affects these interactions.

How do ASK1 antibodies compare with newer therapeutic antibodies like MDX-124 in cancer research?

ASK1 antibodies and therapeutic antibodies like MDX-124 serve distinct research purposes:

• Research-grade ASK1 antibodies:

  • Primary use: Detection and analysis of ASK1 protein/phosphorylation

  • Applications: Western blotting, IHC, ELISA, immunoprecipitation

  • Target accessibility: Primarily useful for detecting intracellular ASK1 in fixed or permeabilized samples

  • Research value: Elucidate signaling mechanisms and expression patterns

• Therapeutic antibodies (e.g., MDX-124 targeting ANXA1):

  • Primary use: Functional modulation of target proteins for therapeutic effect

  • Applications: In vitro treatment of cell lines, in vivo treatment of animal models

  • Target design: Engineered to target accessible epitopes on membrane or secreted proteins

  • Research value: Demonstrate target validation and therapeutic potential

• Comparative analysis:

  • ASK1 antibodies primarily serve diagnostic/analytical purposes

  • Therapeutic antibodies like MDX-124 demonstrate mechanisms of action and efficacy

  • MDX-124 has shown anti-proliferative effects through G1 cell cycle arrest and tumor inhibition
    Both types of antibodies provide complementary insights in cancer research programs.

What considerations are important when designing experiments to study ASK1 phosphorylation in cancer progression models?

When designing experiments to study ASK1 phosphorylation in cancer progression:

• Model selection:

  • Choose cell lines with detectable ASK1 expression

  • Consider both high and low aggressive cancer models

  • Include normal cell counterparts as controls

• Stimulus parameters:

  • Define physiologically relevant stressors (oxidative stress, cytokines)

  • Determine appropriate dose-response relationships

  • Establish meaningful time courses (minutes to hours)

• Multi-dimensional analysis:

  • Assess multiple phosphorylation sites simultaneously

  • Correlate phosphorylation with downstream signaling activation

  • Link molecular changes to functional outcomes (proliferation, apoptosis, migration)

• In vivo relevance:

  • Compare findings in cell lines to primary patient samples

  • Consider tissue-specific contexts that may alter ASK1 signaling

  • Develop appropriate animal models to validate findings

• Therapeutic implications:

  • Test how modulating ASK1 phosphorylation affects response to standard therapies

  • Consider combination approaches targeting ASK1 signaling pathways
    These experimental design considerations enhance the translational relevance of ASK1 phosphorylation studies in cancer research.

How can ASK1 antibodies be integrated with emerging AI-driven antibody design technologies?

Integration of ASK1 antibodies with AI-driven antibody design represents an emerging frontier:

• Computational epitope mapping:

  • AI algorithms can predict optimal epitopes on ASK1 for antibody recognition

  • These predictions can guide selection of commercial antibodies or design of new ones

  • Models like RFdiffusion generate antibody blueprints that bind user-specified targets

• Enhanced antibody specificity:

  • AI design tools like RFdiffusion can help develop antibodies with improved specificity for particular ASK1 phosphorylation sites

  • These tools are being trained to design complete human-like antibodies (e.g., scFvs)

  • Such approaches may overcome cross-reactivity challenges with current phospho-antibodies

• Functional antibody development:

  • Beyond detection, AI-designed antibodies could modulate ASK1 function

  • This parallels therapeutic antibody development like MDX-124 for ANXA1

  • AI-designed antibodies could target specific conformations of phosphorylated ASK1

• Experimental validation requirements:

  • AI-designed antibodies still require rigorous experimental validation

  • This includes confirming binding specificity, sensitivity, and performance across applications

  • Validation should follow established protocols for antibody characterization
    This integration promises more precise tools for studying ASK1 biology in complex disease settings.

What are the future directions for ASK1 antibody research in cancer biology?

Future directions for ASK1 antibody research in cancer biology include:

• Development of more specific and sensitive phospho-ASK1 antibodies:

  • Antibodies with improved specificity for individual phosphorylation sites

  • Development of conformation-specific antibodies that recognize activated ASK1

  • Creation of antibodies that can distinguish between different ASK1 complexes

• Integration with advanced imaging technologies:

  • Super-resolution microscopy applications to visualize ASK1 signaling complexes

  • Intravital imaging to track ASK1 activation in live tumor models

  • Multiplexed imaging to simultaneously visualize multiple components of ASK1 pathways

• Therapeutic targeting approaches:

  • Development of function-modulating antibodies against ASK1, similar to therapeutic antibodies like MDX-124

  • Exploration of ASK1 as a biomarker for cancer therapy response

  • Investigation of combined targeting of ASK1 with other stress response pathways

• AI-enhanced antibody design:

  • Utilization of platforms like RFdiffusion to create novel ASK1-targeting antibodies

  • Prediction of optimal antibody properties for specific research applications

  • Development of antibodies that can recognize subtle conformational changes in ASK1
    These advances will improve our understanding of ASK1's role in cancer and potentially lead to new therapeutic strategies.

How should researchers evaluate the quality and reliability of commercially available ASK1 antibodies?

When evaluating commercial ASK1 antibodies, researchers should consider:

• Validation documentation:

  • Check for validation data across multiple applications (WB, IHC, IP)

  • Look for validation in knockout/knockdown systems

  • Assess data from multiple cell types or tissues

  • Verify the antibody has been tested on relevant species (human, mouse, rat)

• Publication record:

  • Search for peer-reviewed publications using the specific antibody

  • Evaluate methods sections for detailed protocols

  • Assess reproducibility across multiple research groups

• Technical specifications:

  • Confirm the immunogen used matches your research needs

  • Verify the antibody format is appropriate (monoclonal vs. polyclonal)

  • Check cross-reactivity profiles, especially for phospho-specific antibodies

• Independent validation:

  • Plan for in-house validation experiments

  • Consider testing multiple antibodies from different vendors

  • Document batch-to-batch variability for critical experiments This systematic evaluation approach helps ensure reliable results in ASK1-focused research programs.