CASP4 Antibody

What is the basic structure and function of Caspase-4 (CASP4)?

Caspase-4 is a cysteine-aspartic acid protease encoded by the CASP4 gene in humans. It is synthesized as a zymogen (inactive precursor) that undergoes cleavage into two key subunits: a 20 kDa (p20) and a 10 kDa (p10) subunit. The active enzyme consists of two heterodimers containing these p20 and p10 subunits. The protein contains a catalytic domain with an active site spanning both the p20 and p10 subunits, along with a non-catalytic Caspase Activation and Recruitment Domain (CARD). Functionally, Caspase-4 can cleave and activate its own precursor protein as well as Caspase-1 precursor, playing a crucial role in inflammatory processes .

How does CASP4 differ between mouse and human models?

While Caspase-4 is encoded by the CASP4 gene in humans, mice express murine Caspase-4 with specific molecular differences. Anti-Caspase-4 antibodies must be validated for specific species reactivity, as antibodies may exhibit different binding affinities between species. For example, some antibodies are validated on mouse tissue and recommended for use with materials from both rodent and human tissues . The mouse Caspase-4 protein has an accession number of P70343 and gene ID of 12363, with synonyms including ICE(rel)II, ICH2, TX, and Protease ICH-2 . When designing cross-species experiments, researchers should carefully verify the antibody's specificity for the target species to ensure reliable results.

What are the main post-translational modifications of CASP4 and how do they affect antibody recognition?

Caspase-4 undergoes several post-translational modifications that can significantly impact antibody recognition and experimental outcomes:

These modifications can mask epitopes, alter protein conformation, or directly interfere with antibody binding. When selecting antibodies for modified CASP4 detection, researchers should consider antibodies raised against specific modified epitopes, such as the Cleaved-Caspase 4 (Gln81) antibody, which specifically recognizes the cleaved form .

What are the critical considerations when selecting a CASP4 antibody for specific applications?

When selecting a CASP4 antibody for research, consider:

• Application compatibility: Verify the antibody has been validated for your intended application. For instance, certain CASP4 antibodies are validated for western blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), and immunoprecipitation (IP) .

• Species reactivity: Confirm the antibody recognizes CASP4 in your species of interest. Some antibodies react with human CASP4, while others react with mouse or have cross-reactivity across species .

• Epitope specificity: Determine whether you need an antibody that recognizes the full-length protein, specific domains (like CARD), or cleaved/activated forms. For example, the Cleaved-Caspase 4 (Gln81) Antibody specifically detects the cleaved form at Gln81 .

• Clonality: Polyclonal antibodies offer broader epitope recognition but may have more batch-to-batch variation, while monoclonal antibodies provide consistent specificity to a single epitope.

• Validation data: Review published literature and manufacturer validation data showing the antibody's performance in your specific application and model system.

How should I optimize immunohistochemistry protocols for CASP4 detection in tumor samples?

Optimizing IHC protocols for CASP4 detection in tumor samples requires methodical adjustment of multiple parameters:

• Tissue preparation and fixation:

  • Use 10% neutral buffered formalin fixation for 24-48 hours

  • Ensure proper tissue dehydration and paraffin embedding

  • Cut sections at 4-5 μm thickness for optimal antibody penetration

• Antigen retrieval:

  • Test both heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0)

  • For CASP4, heat-induced retrieval at pH 9.0 often yields better results for detecting cleaved forms

• Antibody dilution and incubation:

  • Perform dilution series (typically 1:100 to 1:1000) to determine optimal concentration

  • Test both overnight incubation at 4°C and 1-hour incubation at room temperature

  • Include appropriate positive controls (e.g., lung adenocarcinoma tissue sections known to express CASP4)

• Detection system:

  • For weak signals, use amplification systems like tyramide signal amplification

  • For co-localization studies, employ fluorescent secondary antibodies optimized for multiplexing

• Controls:

  • Include CASP4 knockout tissues or CASP4-negative cell lines as negative controls

  • Use tissues with confirmed CASP4 expression as positive controls

  • Employ isotype controls to assess non-specific binding

What are the best approaches for detecting both precursor and cleaved forms of CASP4 in western blots?

To effectively detect both precursor and cleaved forms of CASP4 in western blots:

• Sample preparation:

  • Extract proteins using lysis buffers containing protease inhibitors to prevent artificial cleavage

  • Include phosphatase inhibitors to preserve phosphorylation states

  • Process samples quickly and keep them cold to minimize degradation

• Gel selection and separation:

  • Use gradient gels (e.g., 4-20%) to optimally separate both the full-length (≈43 kDa) and cleaved forms (≈20 kDa and ≈10 kDa)

  • Consider using Tricine-SDS-PAGE for better resolution of smaller fragments

• Antibody selection:

  • Utilize antibodies that recognize the pro-domain for detecting precursor forms

  • Use cleaved-specific antibodies like Cleaved-Caspase 4 (Gln81) for activated forms

  • For comprehensive detection, consider antibodies targeting conserved regions present in both forms

• Sequential or dual probing strategies:

  • Strip and reprobe membranes with different CASP4 antibodies

  • Use dual-color detection with antibodies raised in different species

• Positive controls:

  • Include recombinant CASP4 in both full-length and cleaved forms

  • Use cellular lysates treated with lipopolysaccharide (LPS) or endoplasmic reticulum stress inducers to generate cleaved forms

How is CASP4 involved in cancer progression and metastasis?

Recent research has revealed complex roles for CASP4 in cancer biology, particularly in lung adenocarcinoma:

• Pro-metastatic functions:

  • Elevated CASP4 expression in primary lung tumors is associated with cancer progression in patients

  • CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models

  • CASP4 enhances expression of genes associated with angiogenesis and cell migration through nuclear factor kappa-light chain-enhancer of activated B cell signaling, independent of lipopolysaccharide or tumor necrosis factor stimulation

• Dual role in tumor biology:

  • CASP4 contributes to both tumor progression (via angiogenesis and tumor hyperkinesis) and tumor cell killing (in response to high interferon-γ levels)

  • Lung adenocarcinoma cells with high CASP4 expression are more susceptible to interferon-γ-induced pyroptosis than those with low expression

  • CASP4 levels in primary lung adenocarcinoma may predict both metastasis potential and responsiveness to high-dose interferon-γ therapy due to cancer cell pyroptosis

• Induction pathways:

  • CASP4 is induced by endoplasmic reticulum stress or interferon-γ via signal transducer and activator of transcription 1 (STAT1)

  • This suggests potential therapeutic strategies targeting these induction pathways

What are the challenges in interpreting CASP4 expression data in inflammatory versus cancer contexts?

Interpreting CASP4 expression data presents several key challenges:

• Context-dependent functions:

  • In inflammatory contexts, CASP4 primarily serves as a mediator of pyroptosis and inflammatory responses

  • In cancer contexts, CASP4 exhibits dual functionality promoting both tumor progression and potential tumor cell death

  • These seemingly contradictory roles require careful experimental design to distinguish

• Technical considerations:

  • Antibody specificity for different CASP4 activation states may lead to inconsistent results across studies

  • Expression levels versus activation status - high expression doesn't necessarily correlate with high activity

  • Subcellular localization affects function but may be overlooked in total expression studies

• Methodological approach comparisons:

  • RNA expression (qPCR, RNA-seq) versus protein expression (western blot, IHC) may yield discordant results

  • Single-cell versus bulk tissue analysis can reveal different patterns due to heterogeneous expression across cell populations

  • In situ detection versus lysate-based methods may highlight different aspects of CASP4 biology

• Cross-talk with other inflammatory pathways:

  • CASP4 interacts with multiple inflammatory pathways including NF-κB signaling

  • Distinguishing CASP4-specific effects from broader inflammatory responses requires specific inhibitors or genetic models

How can researchers effectively study CASP4-mediated pyroptosis in cancer treatment response models?

To effectively study CASP4-mediated pyroptosis in cancer treatment response models, researchers should consider:

• Experimental model selection:

  • Cell line models with varying CASP4 expression levels (e.g., lung adenocarcinoma cell lines)

  • Patient-derived xenografts to maintain tumor heterogeneity

  • Genetic models with inducible CASP4 expression or knockout

  • 3D organoid cultures to better recapitulate tissue architecture

• Pyroptosis detection methods:

  • Membrane integrity assays (LDH release, propidium iodide uptake)

  • Analysis of pyroptosis-specific morphological changes using time-lapse microscopy

  • Detection of cleaved gasdermin D (the pyroptosis executioner protein)

  • Measurement of released inflammatory cytokines (IL-1β, IL-18)

• Therapeutic context design:

  • Combination studies with interferon-γ treatment at varying concentrations to identify thresholds for pyroptosis induction

  • Time-course experiments to distinguish early versus late pyroptotic events

  • Comparison studies between conventional therapies and pyroptosis-inducing approaches

• Mechanistic investigations:

  • Use of specific CASP4 inhibitors versus genetic knockdown/knockout to distinguish between enzymatic and scaffolding functions

  • Investigation of upstream activators in the cancer context, including endoplasmic reticulum stress

  • Analysis of CASP4 induction via STAT1 signaling following interferon-γ treatment

• Translational correlates:

  • Correlation of in vitro/in vivo pyroptosis susceptibility with CASP4 expression levels

  • Development of biomarkers that predict response to pyroptosis-inducing therapies

  • Analysis of immune cell recruitment following pyroptotic cell death

How do I troubleshoot non-specific binding or weak signals when using CASP4 antibodies?

When encountering non-specific binding or weak signals with CASP4 antibodies, implement this systematic troubleshooting approach:

• For non-specific binding issues:

  • Increase blocking stringency using 5% BSA or 5% milk in TBST

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Perform additional washing steps with higher salt concentration (up to 500 mM NaCl)

  • Use more specific antibodies like those targeting cleaved forms at specific residues (e.g., Cleaved-Caspase 4 at Gln81)

  • Include competing peptides corresponding to the antibody epitope to verify specificity

  • Use tissues or cells from CASP4 knockout models as negative controls

• For weak signal problems:

  • Optimize protein extraction using different lysis buffers to ensure complete protein recovery

  • Reduce protease activity during sample preparation by adding additional protease inhibitors

  • Increase antibody concentration incrementally (typically 2-fold increases)

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

  • Use signal amplification systems like biotin-streptavidin or tyramide signal amplification

  • Try alternative antigen retrieval methods for IHC applications

• Confirming antibody performance:

  • Use positive control samples with known CASP4 expression levels

  • Try multiple antibodies targeting different epitopes of CASP4

  • Verify antibody lot-to-lot consistency with standardized positive controls

What are the optimal conditions for studying CASP4 activation in response to endoplasmic reticulum stress?

Studying CASP4 activation in response to endoplasmic reticulum (ER) stress requires careful experimental design:

• ER stress induction protocols:

  • Pharmacological inducers: tunicamycin (1-5 μg/mL, 6-24h), thapsigargin (0.1-1 μM, 4-16h), or brefeldin A (1-5 μg/mL, 6-24h)

  • Physiological stressors: glucose deprivation, hypoxia (1% O₂), or calcium dysregulation

  • Optimization of concentration and time course is critical as excessive ER stress leads to apoptosis rather than specific CASP4 activation

• CASP4 activation assessment:

  • Western blot analysis of cleaved CASP4 using cleaved-specific antibodies

  • Enzymatic activity assays using CASP4-specific fluorogenic substrates

  • Co-immunoprecipitation studies to detect CASP4 interaction with activating platforms

  • Subcellular fractionation to track CASP4 translocation during activation

• Molecular pathway verification:

  • Monitor canonical ER stress markers (BiP/GRP78, CHOP, XBP1 splicing) alongside CASP4

  • Use pharmacological inhibitors of specific ER stress pathways (PERK, IRE1α, ATF6) to dissect contribution to CASP4 activation

  • Employ siRNA/shRNA against key ER stress mediators to confirm pathway involvement

• Downstream signaling analysis:

  • Measure NF-κB activation using reporter assays or phospho-IκB detection

  • Analyze cytokine production profiles (IL-1β, IL-18) as functional readouts

  • Assess pyroptosis markers including gasdermin D cleavage and membrane integrity

How can multiplex immunofluorescence be optimized for studying CASP4 interactions with other inflammatory pathway components?

Optimizing multiplex immunofluorescence for CASP4 and inflammatory pathway components requires addressing several technical challenges:

• Antibody panel design:

  • Select primary antibodies from different host species (rabbit anti-CASP4 , mouse anti-inflammasome components, goat anti-cytokines)

  • Validate antibodies individually before multiplexing to establish optimal conditions

  • Consider using directly conjugated primary antibodies to eliminate cross-reactivity

  • Include isotype controls for each species to assess non-specific binding

• Sequential staining approaches:

  • Implement tyramide signal amplification (TSA) with sequential antibody stripping

  • Optimize order of antibody application (typically from weakest to strongest signal)

  • Validate complete stripping between rounds using secondary-only controls

  • Consider spectral unmixing systems for closely overlapping fluorophores

• Sample preparation optimization:

  • Test multiple fixation protocols to preserve both CASP4 and partner proteins

  • Optimize antigen retrieval conditions compatible with all targets

  • Employ background reduction techniques (Sudan Black B, TrueBlack, or autofluorescence quenching reagents)

• Analysis and quantification strategies:

  • Use computational approaches for colocalization analysis (Pearson's correlation, Manders' overlap)

  • Implement cell segmentation algorithms to assess cellular heterogeneity

  • Develop quantitative metrics for activation states (nuclear translocation, aggregation)

  • Consider machine learning approaches for pattern recognition in complex datasets

• Controls for result validation:

  • Include single-color controls for spectral overlap correction

  • Use biological controls with known interaction patterns

  • Confirm key findings with complementary techniques (proximity ligation assay, co-immunoprecipitation)

What is the potential for CASP4 expression as a biomarker for cancer treatment response?

Recent research suggests significant potential for CASP4 as a biomarker for cancer treatment response:

• Predictive value in immunotherapy response:

  • CASP4 expression levels in primary lung adenocarcinoma may predict responsiveness to high-dose interferon-γ therapy

  • High CASP4-expressing cancer cells show increased susceptibility to pyroptosis, a potentially immunogenic form of cell death

  • CASP4 expression could potentially stratify patients for immunotherapies that rely on inflammatory cell death mechanisms

• Metastasis prediction capabilities:

  • Elevated CASP4 expression in primary tumors correlates with cancer progression in lung adenocarcinoma patients

  • Expression patterns may help identify patients at higher risk for metastatic spread

  • This could inform more aggressive treatment approaches for high-risk patients

• Technical considerations for biomarker development:

  • Standardization of CASP4 detection methods across clinical laboratories

  • Establishment of expression thresholds that correlate with treatment outcomes

  • Integration with other inflammatory biomarkers for improved predictive power

• Challenges in implementation:

  • Distinguishing between expression levels and activation status

  • Accounting for tumor heterogeneity in expression patterns

  • Validation across diverse patient populations and cancer types

How does the interaction between CASP4 and angiogenesis pathways influence tumor progression?

The interaction between CASP4 and angiogenesis represents a complex relationship with significant implications for tumor biology:

• Molecular mechanisms linking CASP4 to angiogenesis:

  • CASP4 enhances expression of genes associated with angiogenesis through NF-κB signaling

  • This occurs independently of traditional inflammatory triggers like lipopolysaccharide or tumor necrosis factor

  • CASP4 knockout attenuates tumor angiogenesis in subcutaneous tumor mouse models

• Potential signaling pathways:

  • Direct or indirect activation of key angiogenic factors (VEGF, bFGF, angiopoietins)

  • Modulation of endothelial cell response to angiogenic stimuli

  • Influence on extracellular matrix remodeling to facilitate vessel formation

• Therapeutic implications:

  • CASP4 inhibition could potentially reduce tumor angiogenesis

  • Combining anti-angiogenic therapies with CASP4 modulation might enhance efficacy

  • Selective targeting of CASP4 in cancer cells versus stromal cells may produce different outcomes

• Research approaches to further explore this connection:

  • Endothelial-specific CASP4 manipulation to distinguish direct versus indirect effects

  • 3D co-culture models with tumor cells and endothelial cells to study dynamic interactions

  • In vivo imaging of tumor vasculature in CASP4-modified models

What are the methodological considerations for developing selective CASP4 inhibitors for research applications?

Developing selective CASP4 inhibitors for research requires addressing several methodological considerations:

• Target site selection strategies:

  • Catalytic site targeting: Design substrate-mimetic inhibitors that interact with the active site spanning the p20 and p10 subunits

  • Allosteric site targeting: Identify regulatory domains that could modulate activity without competing with substrates

  • CARD domain targeting: Develop inhibitors that disrupt protein-protein interactions necessary for activation

• Selectivity challenges and approaches:

  • Address high homology between inflammatory caspases (CASP1, CASP4, CASP5)

  • Exploit subtle differences in substrate binding pockets

  • Use structure-based design informed by crystal structures

  • Implement activity-based protein profiling to assess selectivity across the caspase family

• Validation methodologies:

  • Enzymatic assays using recombinant proteins to determine IC₅₀ values

  • Cellular assays monitoring CASP4-dependent functions (pyroptosis, IL-1β processing)

  • Confirmatory studies comparing inhibitor effects with genetic knockdown/knockout models

  • Pharmacokinetic and biodistribution studies for in vivo applications

• Delivery systems for effective inhibition:

  • Cell-penetrating peptide conjugation for intracellular delivery

  • Nanoparticle encapsulation for targeted delivery to specific tissues

  • Prodrug approaches for improved stability and bioavailability

  • Inducible expression systems for conditional inhibition in genetic models