Cleaved-CASP5 (D121) Antibody

What is Cleaved-CASP5 (D121) Antibody and what does it detect?

Cleaved-CASP5 (D121) Antibody is a rabbit polyclonal antibody specifically designed to detect endogenous levels of activated Caspase-5 p20 protein fragments resulting from cleavage adjacent to aspartic acid at position 121 (D121). This antibody recognizes the cleaved form of Caspase-5, which occurs during activation of the protein in inflammatory and cell death pathways. It does not recognize the full-length, inactive form of the protein, making it valuable for studying Caspase-5 activation events .

What is the biological function of Caspase-5?

Caspase-5 is a thiol protease that functions as a mediator of programmed cell death. It plays a critical role in:

• Initiating pyroptosis, a programmed lytic cell death pathway, through cleavage of Gasdermin-D (GSDMD)

• Releasing the N-terminal gasdermin moiety that binds to membranes and forms pores, triggering pyroptosis

• Mediating cleavage and maturation of inflammatory cytokines such as IL-18

• Potentially regulating antiviral innate immune activation during non-canonical inflammasome activation by cleaving CGAS

Understanding Caspase-5 activation is essential for research into inflammatory processes, cell death mechanisms, and innate immunity.

What applications is the Cleaved-CASP5 (D121) Antibody validated for?

The antibody has been validated for the following research applications:

It has been specifically tested and confirmed to work with human samples, with some vendors also claiming reactivity with rat and mouse samples .

How can I distinguish between different cleaved forms of Caspase-5 in my experiments?

Distinguishing between different cleaved forms of Caspase-5 requires careful experimental design:

• Antibody selection: Use site-specific antibodies like the Cleaved-CASP5 (D121) Antibody that target specific cleavage sites. For comprehensive analysis, consider using antibodies against different cleavage sites (D121 vs. Ser331) or domains (p10 vs. p20) .

• Molecular weight analysis: Different cleavage sites generate fragments of distinct molecular weights. In Western blots, the p20 subunit (containing the D121 cleavage site) appears at approximately 20 kDa, while the p10 subunit appears at approximately 10 kDa.

• Positive controls: Include lysates from cells with known Caspase-5 activation, such as HeLa cells treated with inflammasome activators .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm specificity of detection.

• Mass spectrometry: For definitive identification of specific cleavage products, consider immunoprecipitation followed by mass spectrometry (IP-MS) approaches similar to those used for other inflammatory caspases .

How does the mechanism of caspase cleavage detection by antibodies like Cleaved-CASP5 (D121) compare to broader caspase cleavage motif antibodies?

The mechanism of detection differs significantly:

• Site-specific antibodies (like Cleaved-CASP5 D121):

  • Recognize a specific neo-epitope created after cleavage at a precise amino acid position

  • Highly specific for a single cleavage event in one protein

  • Useful for monitoring activation of specific caspases

  • Limited to detecting known cleavage events

• Cleavage motif antibodies (like CJ2 and CJ11 described in search result ):

  • Recognize a degenerate cleavage motif pattern common to multiple substrates

  • Can detect numerous different proteins cleaved by the same caspase

  • Enable discovery of novel substrates through immunoprecipitation-mass spectrometry approaches

  • Particularly valuable for pathway analysis

For example, the CJ11 antibody described in the literature can selectively immunoprecipitate the cleaved forms of multiple inflammatory caspase substrates, including IL-1β, IL-18, caspase-11, and GSDMD. This broader specificity allows for discovery of hundreds of putative substrates of the noncanonical inflammasome .

When designing experiments to study Caspase-5 function, researchers should consider whether they need to monitor a specific cleavage event (use Cleaved-CASP5 D121) or discover novel substrates (use a motif-specific antibody).

What is the relationship between Caspase-5 activation and inflammasome function?

Caspase-5 is an inflammatory caspase that plays distinct roles in inflammasome signaling:

• Noncanonical inflammasome pathway: Caspase-5 can initiate pyroptosis through cleavage of Gasdermin-D (GSDMD), releasing the N-terminal fragment that forms membrane pores.

• Cytokine processing: Caspase-5 mediates cleavage and maturation of IL-18, contributing to inflammatory responses.

• Exosite-dependent substrate recognition: The cleavage of GSDMD and IL-18 by Caspase-5 is not strictly dependent on the consensus cleavage site but depends on an exosite interface similar to that described for CASP4.

• Antiviral response: During non-canonical inflammasome activation, Caspase-5 cuts CGAS and may play a role in regulating antiviral innate immune activation .

Understanding these relationships is critical when designing experiments to study inflammasome biology. When using the Cleaved-CASP5 (D121) Antibody, researchers should consider additional markers of inflammasome activation (such as cleaved Gasdermin-D or mature IL-1β) to comprehensively assess pathway activity.

What are the optimal conditions for using Cleaved-CASP5 (D121) Antibody in Western blotting?

For optimal Western blot results with Cleaved-CASP5 (D121) Antibody:

• Sample preparation:

  • Lyse cells in buffer containing protease inhibitors to prevent artificial cleavage

  • Include positive controls (e.g., HeLa cells with inflammasome activation)

  • Consider enriching for cleaved proteins through immunoprecipitation

• Protein loading and transfer:

  • Load 20-50 μg of total protein per lane

  • Use PVDF membranes for optimal protein binding

  • Transfer at 100V for 60-90 minutes or 30V overnight at 4°C

• Antibody dilution and incubation:

  • Dilute primary antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C for optimal binding

  • Use a compatible HRP-conjugated secondary anti-rabbit antibody at 1:5000-1:10000

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrates

  • Exposure times may need optimization based on signal strength

  • Consider digital imaging systems for quantitative analysis

• Blocking and washing:

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

  • Perform at least 3-5 washes with TBST after each antibody incubation

How can I induce and detect Caspase-5 cleavage in cell culture models?

To effectively induce and detect Caspase-5 cleavage:

• Cell line selection:

  • HeLa cells have been validated for Cleaved-CASP5 (D121) detection

  • Primary human monocytes or macrophages express higher levels of Caspase-5

  • Consider cell lines with known inflammasome components

• Induction protocols:

  • LPS priming (100 ng/ml, 4-6 hours) followed by ATP (5 mM, 30 minutes)

  • Cytosolic bacterial delivery using transfection reagents

  • Bacterial infection with inflammasome-activating pathogens like Legionella pneumophila

• Detection methods:

  • Western blot using Cleaved-CASP5 (D121) Antibody (1:500-1:2000)

  • Immunofluorescence microscopy to visualize cellular localization

  • Flow cytometry for population-level analysis

  • Combine with assays for downstream effects (LDH release, IL-1β ELISA)

• Controls:

  • Include caspase inhibitors (e.g., Z-VAD-FMK) as negative controls

  • Use known inflammasome activators as positive controls

  • Consider genetic knockdown/knockout validation

What are the best methods for preserving Caspase-5 cleavage products in tissue samples?

Preserving Caspase-5 cleavage products in tissues requires careful handling:

• Tissue collection:

  • Collect samples rapidly after sacrifice/biopsy

  • Immediately flash-freeze in liquid nitrogen or preserve in appropriate fixative

  • Consider using caspase inhibitors in collection buffers

• Fixation options:

  • For immunohistochemistry: 10% neutral buffered formalin, 24 hours

  • For immunofluorescence: 4% paraformaldehyde, 4-6 hours

  • For frozen sections: OCT embedding after brief 4% PFA fixation

• Protein extraction for biochemical analysis:

  • Homogenize tissues in RIPA buffer with protease inhibitors at 4°C

  • Centrifuge at high speed (14,000 × g) to remove debris

  • Process immediately or store at -80°C in single-use aliquots

  • Avoid repeated freeze-thaw cycles

• Antibody validation:

  • Optimize antibody concentration for each tissue type

  • Include appropriate positive and negative controls

  • Consider antigen retrieval methods for fixed tissues

Why might I see multiple bands when using the Cleaved-CASP5 (D121) Antibody in Western blot?

Multiple bands in Western blots can occur for several reasons:

• Expected multiple fragments:

  • The active form of Caspase-5 consists of a heterotetramer with two p20 and two p10 subunits

  • You may detect both the p20 fragment (~20 kDa) containing the D121 cleavage site and intermediate cleavage products

• Non-specific binding:

  • Try increasing the dilution of primary antibody (1:1000-1:2000)

  • Optimize blocking conditions (5% BSA may reduce background compared to milk)

  • Increase washing steps and duration (5 × 5 minutes with TBST)

• Cross-reactivity with other caspases:

  • Inflammatory caspases share sequence homology

  • Perform peptide competition assays to confirm specificity

  • Include samples from Caspase-5 knockout cells as negative controls

• Post-translational modifications:

  • Phosphorylation or other modifications may alter migration

  • Consider phosphatase treatment of lysates if suspected

• Sample degradation:

  • Always use fresh samples or those stored properly at -80°C

  • Include protease inhibitors in all buffers

  • Avoid repeated freeze-thaw cycles

How should I interpret changes in Cleaved-CASP5 (D121) levels in experimental models of inflammation?

Interpretation of Cleaved-CASP5 (D121) levels requires careful consideration:

• Time course analysis:

  • Caspase activation is often transient

  • Perform time course experiments to capture peak activation

  • Compare with kinetics of upstream triggers and downstream effects

• Correlation with functional outcomes:

  • Measure pyroptosis markers (LDH release, propidium iodide uptake)

  • Assess IL-18 maturation and release

  • Evaluate GSDMD cleavage as a downstream target

• Integration with other inflammatory markers:

  • Compare with other inflammasome components (NLRP3, ASC)

  • Assess NF-κB pathway activation (p65 phosphorylation, IκBα degradation)

  • Measure cytokine production patterns

• Consideration of cell-type specific effects:

  • Some cell types (mast cells, neurons) express low GSDMD levels, which may alter outcomes

  • Different cell types may have varying levels of Caspase-5 expression

  • Tissue-specific regulation of inflammasome components can influence results

How can I validate the specificity of the Cleaved-CASP5 (D121) Antibody in my experimental system?

To thoroughly validate antibody specificity:

• Genetic approaches:

  • Use CRISPR/Cas9 to generate Caspase-5 knockout cells

  • Compare wild-type vs. knockout samples

  • Perform rescue experiments with wild-type vs. D121A mutant Caspase-5

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Should abolish specific signal while leaving non-specific signals unchanged

  • Use titrated amounts of competing peptide (similar to the BSA-S672-691 peptide competition approach described in result )

• Multiple detection methods:

  • Compare results across Western blot, immunofluorescence, and ELISA

  • Consistency across methods supports specificity

  • Discrepancies may reveal context-dependent artifacts

• Correlation with known activators/inhibitors:

  • Known inflammasome activators should increase signal

  • Caspase inhibitors should decrease signal

  • Knockdown of upstream regulators should modulate signal

• Mass spectrometry validation:

  • Immunoprecipitate with the Cleaved-CASP5 (D121) Antibody

  • Confirm identity of pulled-down proteins by mass spectrometry

  • Similar to the approach used with the CJ11 antibody in result

How can Cleaved-CASP5 (D121) Antibody be used to study the noncanonical inflammasome pathway?

The antibody offers several approaches to study the noncanonical inflammasome:

• Pathway activation analysis:

  • Monitor Caspase-5 cleavage as a readout of noncanonical inflammasome activation

  • Compare with canonical inflammasome markers

  • Correlate with upstream sensors and downstream effectors

• Substrate identification:

  • Combine with immunoprecipitation and mass spectrometry

  • Identify novel substrates cleaved following Caspase-5 activation

  • Validate findings using recombinant protein cleavage assays

• Regulation studies:

  • Investigate factors that modulate Caspase-5 cleavage

  • Study post-translational modifications affecting cleavage

  • Examine protein-protein interactions influencing activation

• Real-time activation monitoring:

  • Develop cell-based reporters using Cleaved-CASP5 (D121) epitope

  • Create FRET-based sensors for live-cell imaging

  • Adapt for high-throughput screening applications

What are potential applications of Cleaved-CASP5 (D121) Antibody in studying infectious disease mechanisms?

The antibody has valuable applications in infectious disease research:

• Pathogen-induced inflammasome activation:

  • Study how different bacteria activate Caspase-5

  • Compare responses across pathogen species and strains

  • Investigate virulence factors that modulate activation

• Host defense mechanisms:

  • Examine Caspase-5 activation in response to bacterial, viral, or fungal components

  • Assess role in controlling pathogen replication

  • Study integration with other innate immune pathways

• Evasion strategies:

  • Identify pathogen factors that inhibit Caspase-5 activation

  • Similar to how cleaved immunoglobulins are recognized by immune receptors as described in result

  • Compare successful vs. unsuccessful pathogens

• Therapeutic intervention points:

  • Screen for compounds that modulate Caspase-5 activation

  • Identify pathways that can be targeted pharmacologically

  • Develop biomarkers for inflammasome activation in infection

How might Cleaved-CASP5 (D121) Antibody contribute to biomarker development for inflammatory conditions?

The antibody shows potential for biomarker development:

• Tissue-based diagnostics:

  • Assess Caspase-5 activation in patient biopsies

  • Compare levels across disease states and healthy controls

  • Correlate with disease severity and outcomes

• Liquid biopsy approaches:

  • Detect Caspase-5 cleavage products in serum or plasma

  • Develop sandwich ELISA using Cleaved-CASP5 (D121) Antibody

  • Evaluate as predictive biomarkers for disease flares

• Therapeutic monitoring:

  • Measure changes in Caspase-5 activation during treatment

  • Assess as pharmacodynamic biomarkers

  • Correlate with clinical responses

• Integration with multi-parameter panels:

  • Combine with other inflammasome activation markers

  • Develop multiplexed assays for comprehensive pathway analysis

  • Use machine learning to identify diagnostic signatures