Cleaved-CASP4/CASP5 (D270/D311) Antibody

What is the specificity of Cleaved-CASP4/CASP5 (D270/D311) Antibody and how was it generated?

The Cleaved-CASP4/CASP5 (D270/D311) Polyclonal Antibody specifically detects endogenous levels of activated Caspase-4/5 p20 protein fragments resulting from cleavage adjacent to D270 (CASP4) and D311 (CASP5). This antibody was generated using a synthesized peptide derived from the internal region of human Caspase 4/5, specifically from amino acids 221-270 . The antibody underwent affinity purification from rabbit antiserum using epitope-specific immunogen chromatography, resulting in high specificity for the cleaved form of these inflammatory caspases .

Unlike antibodies targeting the full-length proteins, this antibody specifically recognizes the activated forms that occur during inflammasome activation and pyroptotic cell death pathways. This specificity makes it particularly valuable for researchers studying caspase activation in inflammatory processes and bacterial infections.

What are the validated applications for this antibody and their recommended protocols?

The Cleaved-CASP4/CASP5 (D270/D311) Antibody has been validated for several research applications with specific recommended protocols:

While not extensively validated, some researchers have also applied this antibody for immunohistochemistry (IHC) and immunofluorescence (IF) with appropriate optimization steps . For these applications, starting dilutions of 1:100-1:300 are recommended, with subsequent optimization for specific tissue types and fixation methods .

What is the optimal storage and handling procedure for maintaining antibody performance?

For optimal antibody performance and stability, follow these storage and handling recommendations:

The antibody should be stored at -20°C for up to 1 year from the date of receipt . The formulation typically includes PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which helps maintain stability during storage .

To maximize antibody performance and shelf life:

• Aliquot the antibody upon receipt to avoid repeated freeze/thaw cycles

• Keep the antibody on ice when in use

• Return to storage at -20°C promptly after use

• Store at -80°C for longer-term storage according to some manufacturers

• Maintain a concentration of 1 mg/ml for stability

Proper storage and handling ensure consistent performance and extend the useful life of this valuable research reagent.

How can I validate the specificity of Cleaved-CASP4/CASP5 (D270/D311) Antibody in my experimental system?

Rigorous validation is essential for ensuring reliable experimental results. For Cleaved-CASP4/CASP5 (D270/D311) Antibody, implement these validation strategies:

• Positive and Negative Controls:

  • Positive control: Treat human monocytes or macrophages with LPS (1-10 μg/ml for 4-6 hours) to induce CASP4/CASP5 activation

  • Negative control: Use CASP4/CASP5 knockout cells or tissues, or samples from sepsis-associated immunosuppression patients where CASP4 is downregulated

• Band Size Verification:

  • The cleaved p20 fragment should appear at approximately 22 kDa on Western blots

  • The full-length protein appears at approximately 47 kDa

  • Both bands may be present in partially activated samples

• siRNA Knockdown:

  • Transfect cells with siRNA targeting CASP4/CASP5

  • Compare signal between knockdown and control samples

  • Expect significant reduction in both full-length and cleaved forms

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide

  • This should block specific binding and eliminate specific signals

This comprehensive validation approach will confirm that the observed signals genuinely represent cleaved CASP4/CASP5 and not non-specific binding or cross-reactivity.

What is the relationship between CASP4/CASP5 activation and GSDMD cleavage, and how can this antibody help study this process?

CASP4/CASP5 directly cleave Gasdermin D (GSDMD) at position D275, which is a critical step in pyroptosis execution . Understanding this relationship is essential for studying inflammatory cell death, and this antibody provides a valuable tool for this research:

• Sequential Detection Approach:

  • Use Western blot to detect both cleaved CASP4/CASP5 (using this antibody) and cleaved GSDMD

  • The temporal relationship helps establish the sequence of events in pyroptosis

  • CASP4/CASP5 activation typically precedes GSDMD cleavage

• Inhibitor Studies:

  • Apply CASP4/CASP5 inhibitors and monitor:

    • Cleaved CASP4/CASP5 levels (should decrease)

    • GSDMD cleavage (should decrease if dependent on CASP4/CASP5)

  • This confirms the causal relationship between caspase activation and GSDMD processing

• Correlation in Clinical Samples:

  • In patients with sepsis-associated immunosuppression, CASP4 downregulation correlates with decreased GSDMD activity

  • This antibody can help establish this correlation in various clinical contexts

This antibody allows researchers to specifically track the activation state of CASP4/CASP5, providing critical insights into how these inflammatory caspases regulate pyroptosis through GSDMD cleavage.

How do CASP4 and CASP5 differ in their regulation during inflammatory conditions, and how can this antibody help distinguish their roles?

Despite their similar functions, CASP4 and CASP5 show distinct regulatory patterns in inflammatory conditions. Recent research has revealed differential regulation during acute-on-chronic liver failure and sepsis-associated immunosuppression :

• Differential Regulation Pattern:

  • CASP5 remains upregulated during critical illness and immunosuppression

  • CASP4 becomes downregulated in patients exhibiting features of immunosuppression and organ failure

  • This suggests non-redundant functions in the inflammasome response to infection

• Mechanistic Differences:

  • CASP4 downregulation correlates with:

    • Decreased gasdermin D levels

    • Impaired interferon signaling

    • Reduced activity of IRF1/IRF2 (CASP4 transcriptional activators)

  • CASP5 regulation appears to follow different pathways

• Clinical Correlations:

  • CASP4 expression inversely correlates with markers of organ dysfunction:

    • MELD score (liver function)

    • SOFA score (multi-organ failure)

  • This establishes CASP4 as a potential biomarker for disease severity

While this antibody recognizes both CASP4 and CASP5 cleaved forms, using it in combination with specific transcriptional analysis can help distinguish their distinct roles in inflammatory diseases and sepsis.

What are the optimal sample preparation methods for detecting cleaved CASP4/CASP5?

Proper sample preparation is crucial for detecting cleaved caspases, which can be relatively unstable. For optimal results with this antibody:

• Cell Lysis Buffer Composition:

  • Use a lysis buffer containing:

    • 50 mM Tris-HCl (pH 7.4)

    • 150 mM NaCl

    • 1% NP-40 or Triton X-100

    • Protease inhibitor cocktail (fresh)

  • Process samples immediately on ice to prevent further processing during lysis

• Protein Preservation for Western Blot:

  • Add reducing agents (DTT or β-mercaptoethanol) to maintain reducing conditions

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Avoid excessive heating (95°C for more than 5 minutes) which may degrade epitopes

• Tissue Sample Processing:

  • For FFPE tissues: Use proper fixation (4% paraformaldehyde for 24-48 hours)

  • For frozen sections: Fix briefly post-sectioning (2% PFA for 10-15 minutes)

  • Apply appropriate antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

These sample preparation methods help preserve the cleaved forms of CASP4/CASP5 and maximize detection sensitivity with this antibody.

What positive controls should be used for experiments with this antibody?

Effective positive controls are essential for interpreting results with confidence. For Cleaved-CASP4/CASP5 (D270/D311) Antibody, consider these positive controls:

• Cell-based Positive Controls:

  • LPS-treated human monocytes or macrophages: Treatment with LPS (1-10 μg/ml for 4-6 hours) activates the non-canonical inflammasome

  • Cytosolic LPS delivery: Transfection of LPS directly into the cytosol using transfection reagents

  • Gram-negative bacterial infection: Infection with bacteria like Salmonella or E. coli

• Tissue-based Positive Controls:

  • Intestinal epithelial tissue: Widely expresses CASP4/CASP5 at the protein level

  • Keratinocytes: Express CASP4 and release it following UVB irradiation

  • Sepsis patient samples: Show differential regulation of CASP4/CASP5

• Known Detection Patterns:

  • Expect to observe bands at approximately 22 kDa (cleaved p20 fragment) and possibly 47 kDa (full-length protein)

  • In activated samples, the ratio of cleaved to full-length protein should increase

These positive controls provide reliable reference points for validating antibody performance and interpreting experimental results across different biological contexts.

How can post-translational modifications affect the detection of cleaved CASP4/CASP5?

Post-translational modifications (PTMs) can significantly impact antibody recognition of cleaved CASP4/CASP5:

• Phosphorylation:

  • CASP4 can be phosphorylated at S271 and S274, very close to the D270 cleavage site

  • This phosphorylation may interfere with antibody binding by altering epitope structure

  • Consider phosphatase treatment of samples if inconsistent results are observed

• ADP-ribosylation:

  • Bacterial pathogens like Shigella flexneri can ADP-ribosylate CASP4 via the effector OspC3

  • This modification blocks CASP4 autoprocessing, preventing activation and GSDMD cleavage

  • This biological mechanism may explain reduced antibody signal in certain infection models

• Ubiquitination:

  • CASP4 has several ubiquitination sites (K87, K107, K129, K225)

  • These modifications may affect protein stability and epitope accessibility

  • Consider using proteasome inhibitors to study ubiquitination effects on cleaved caspase levels

Understanding these potential PTM effects can help interpret experimental results, especially in complex biological systems like infection models or disease tissues.

How can this antibody be used to study inflammasome activation in infectious and inflammatory diseases?

The Cleaved-CASP4/CASP5 (D270/D311) Antibody serves as a valuable tool for studying inflammasome activation in various disease contexts:

• Bacterial Infection Models:

  • Monitor CASP4/CASP5 activation during gram-negative bacterial infections

  • Compare activation patterns between different bacterial species

  • Correlate with bacterial clearance and inflammatory outcomes

  • This helps understand how the non-canonical inflammasome responds to bacterial threats

• Inflammatory Disease Research:

  • Study CASP4/CASP5 activation in models of:

    • Inflammatory bowel disease

    • Acute liver injury

    • Sepsis and septic shock

  • Correlate activation with disease progression and severity markers

• Multi-marker Inflammasome Analysis:

  • Combine detection of cleaved CASP4/CASP5 with:

    • Cleaved CASP1 (canonical inflammasome)

    • ASC speck formation

    • NLRP3 activation

    • IL-1β/IL-18 processing

  • This provides a comprehensive view of inflammasome pathway activation

This antibody enables researchers to specifically track the activation of non-canonical inflammasome components, providing insights into disease mechanisms and potential therapeutic targets.

What insights can be gained from studying CASP4/CASP5 activation in sepsis and immunosuppression?

Recent research has revealed important insights about CASP4/CASP5 in sepsis and immunosuppression that can be further explored using this antibody:

• Differential Regulation in Critical Illness:

  • CASP4 and CASP5 are differentially regulated during acute-on-chronic liver failure and sepsis-associated immunosuppression

  • While CASP5 remains upregulated, CASP4 becomes downregulated specifically in immunosuppressed patients

• Correlation with Clinical Parameters:

  • CASP4 downregulation correlates with:

    • Higher MELD and SOFA scores (markers of organ dysfunction)

    • Decreased GSDMD activity

    • Impaired interferon signaling

  • These correlations suggest CASP4 as a potential biomarker for immunosuppression severity

• Mechanistic Insights:

  • The selective downregulation of CASP4 (but not CASP5) suggests distinct roles in immune function

  • Decreased CASP4 may contribute to impaired inflammatory responses during immunosuppression

  • This antibody can help track these changes at the protein level across different patient populations

These findings provide new directions for developing biomarkers or immunomodulatory therapies for severe infections and sepsis-associated organ damage, with this antibody serving as a key research tool.

How can quantitative analysis of cleaved CASP4/CASP5 be performed in Western blot experiments?

Accurate quantification of CASP4/CASP5 activation provides valuable insights into inflammasome activity. For quantitative analysis using this antibody:

• Normalization Strategy:

  • Normalize cleaved CASP4/CASP5 signal to:

    • Total CASP4/CASP5 (using antibodies against full-length protein)

    • Housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • Calculate the cleaved/total ratio to assess percentage of activation

• Quantification Methodology:

  • Use digital image analysis software (ImageJ, Image Studio)

  • Define regions of interest consistently across all bands

  • Subtract background using adjacent blank areas

  • Use integrated density values rather than peak intensity

• Statistical Analysis Approach:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests based on experimental design

  • Present data as fold-change relative to control conditions

  • Include both cleaved fragment levels and cleaved/total ratios

This quantitative approach provides rigorous assessment of CASP4/CASP5 activation levels, allowing for meaningful comparisons across experimental conditions and clinical samples.

How might this antibody contribute to understanding the non-redundant functions of CASP4 and CASP5?

Despite their structural similarities, emerging evidence suggests CASP4 and CASP5 have non-redundant functions. This antibody can contribute to exploring these differences:

• Differential Expression Studies:

  • Compare CASP4/CASP5 activation patterns across different:

    • Cell types (epithelial cells vs. immune cells)

    • Inflammatory conditions (acute vs. chronic)

    • Disease states (active inflammation vs. immunosuppression)

  • Combine with specific PCR to distinguish between transcriptional and post-translational regulation

• Substrate Specificity Analysis:

  • While both cleave GSDMD at D275, they may have different efficiencies or additional substrates

  • This antibody can help track activation in reconstitution experiments with purified substrates

  • Compare timing and efficiency of substrate processing between CASP4 and CASP5

• Regulation by Post-Translational Modifications:

  • Investigate how different PTMs affect CASP4 vs. CASP5 activation

  • This antibody can help identify differential processing in various cellular contexts

  • May reveal unique regulatory mechanisms for each caspase

This research direction could reveal specialized roles for these inflammatory caspases and potentially identify selective therapeutic targets for various inflammatory conditions.

What methodological advances might improve detection of cleaved CASP4/CASP5 in complex biological samples?

As research on inflammatory caspases advances, several methodological improvements could enhance detection using this antibody:

• Enrichment Techniques:

  • Immunoprecipitation of CASP4/CASP5 before Western blot analysis

  • Subcellular fractionation to concentrate caspases from relevant compartments

  • Proximity ligation assays to detect interaction with substrate proteins

• Advanced Imaging Applications:

  • Super-resolution microscopy to visualize cleaved CASP4/CASP5 localization

  • Live-cell imaging using complementary fluorescent reporter systems

  • Spatial transcriptomics combined with protein detection to map activation patterns

• Single-cell Analysis Methods:

  • Adaptation for flow cytometry to quantify cleaved CASP4/CASP5 at single-cell level

  • Integration with single-cell proteomics for comprehensive pathway analysis

  • Correlation with cell death phenotypes in heterogeneous populations

These methodological advances would extend the utility of this antibody beyond traditional Western blot and ELISA applications, providing deeper insights into the spatial and temporal dynamics of CASP4/CASP5 activation in complex biological systems.