Cleaved-CASP7 (S199) Antibody

What is Cleaved-CASP7 (S199) Antibody and what does it detect?

Cleaved-CASP7 (S199) Antibody is a polyclonal antibody that specifically recognizes the activated form of Caspase-7 protein resulting from cleavage adjacent to Serine 199. This antibody detects endogenous levels of the cleaved fragment of Caspase-7, a critical protease in the apoptotic pathway . It is typically produced against a synthesized peptide derived from human Caspase-7 within amino acid range 180-229 .

The significance of this antibody lies in its ability to detect only the activated form of Caspase-7, making it useful for studying apoptotic signaling pathways. Unlike antibodies that recognize total Caspase-7, the cleaved-specific antibody provides direct evidence of Caspase-7 activation in experimental systems .

How does Caspase-7 differ from other caspases, particularly Caspase-3?

While Caspase-7 was initially considered functionally redundant with Caspase-3 due to shared optimal peptide recognition sequences and common substrates, significant distinctions exist:

Caspase-7 is uniquely activated during inflammation and infection through a NOD-like receptor/caspase-1/caspase-7 cascade, suggesting specialized roles beyond apoptosis execution . This indicates that targeting Caspase-7 specifically may offer different therapeutic possibilities compared to broader caspase inhibition .

What are the recommended applications and dilutions for Cleaved-CASP7 (S199) Antibody?

Based on manufacturer specifications, the following applications and dilutions are recommended:

For optimal results, researchers should perform titration experiments with their specific sample types, as the optimal working concentration may vary depending on experimental conditions and sample source . When using this antibody for the first time, it is advisable to include positive controls such as Jurkat cells treated with apoptosis inducers like etoposide .

What species reactivity can be expected with Cleaved-CASP7 (S199) Antibody?

Most commercial Cleaved-CASP7 (S199) antibodies demonstrate confirmed reactivity with:

• Human (Homo sapiens)

• Mouse (Mus musculus)

Some antibodies may have predicted reactivity (based on sequence homology) with additional species:

• Rat (Rattus norvegicus)

• Pig (Sus scrofa)

• Bovine (Bos taurus)

• Horse (Equus caballus)

• Sheep (Ovis aries)

• Dog (Canis familiaris)

When working with species not explicitly validated, preliminary testing with appropriate positive controls is strongly recommended to confirm cross-reactivity .

What is the significance of detecting cleaved Caspase-7 versus total Caspase-7?

Detecting cleaved Caspase-7 provides direct evidence of caspase activation rather than merely presence. This distinction is crucial because:

• Caspase-7 exists as an inactive proenzyme (35 kDa) until proteolytic processing generates active fragments

• The presence of cleaved Caspase-7 specifically indicates execution phase of apoptosis is underway

• Different proteases (CASP1, CASP8, CASP9, CASP10, GZMB) can activate Caspase-7, leading to distinct programmed cell death processes

• Cleaved Caspase-7 detection allows temporal mapping of apoptotic signaling cascades

• In inflammatory contexts, cleaved Caspase-7 indicates inflammasome activation

Monitoring cleaved versus total Caspase-7 allows researchers to distinguish between cells that merely express the enzyme and those actively undergoing apoptosis or inflammatory responses .

How can I distinguish between Caspase-7 activation by different upstream proteases?

Distinguishing between different Caspase-7 activation pathways requires a strategic experimental approach:

• Specific inhibitor usage: Deploy selective inhibitors for particular upstream caspases (e.g., VX-765 for Caspase-1, Z-IETD-FMK for Caspase-8) alongside Cleaved-CASP7 detection

• Genetic approaches: Utilize siRNA knockdown or CRISPR/Cas9 knockout of specific upstream caspases or inflammasome components (NLRP3, ASC, Ipaf) to determine their contribution to Caspase-7 activation

• Pathway-specific stimulation:

  • For inflammasome/Caspase-1: LPS + ATP stimulation

  • For extrinsic pathway/Caspase-8: Death receptor ligands (TNF-α, FasL)

  • For intrinsic pathway/Caspase-9: DNA-damaging agents (etoposide)

  • For Granzyme B: Purified GzmB or co-culture with cytotoxic T cells

• Time-course experiments: Different activation pathways exhibit distinct temporal dynamics, allowing discrimination based on activation kinetics

• Co-immunoprecipitation: Identify physical association between Caspase-7 and upstream activators

Research has shown that activation of Caspase-7 during bacterial infections like Salmonella specifically requires the inflammasome adaptor ASC and receptors like Ipaf and Cryopyrin, whereas Caspase-3 activation proceeds independently .

What methodological considerations are important when studying Caspase-7 in inflammation versus apoptosis?

When investigating Caspase-7 in different cellular death/stress contexts, researchers should consider:

For inflammation studies:

• Use pathogen-associated molecular patterns (PAMPs) like LPS combined with ATP to trigger inflammasome activation

• Monitor concurrent IL-1β and IL-18 production as markers of inflammasome activity

• Include controls with inflammasome inhibitors (MCC950, Parthenolide)

• Examine Caspase-1 activation in parallel using specific antibodies

• Consider bacterial infection models (e.g., Salmonella) that specifically activate the inflammasome pathway

For apoptosis studies:

• Use classical apoptotic inducers (staurosporine, etoposide, FasL)

• Monitor other apoptotic markers (PARP cleavage, Annexin V staining)

• Examine activation of both Caspase-3 and Caspase-7 to distinguish shared versus unique roles

• Consider pan-caspase inhibitors (Z-VAD-FMK) as controls

Methodological distinctions:

• Sample preparation: Cell lysis buffers should contain appropriate protease inhibitors to prevent ex vivo activation

• Timing: Inflammation-related activation follows different kinetics than classical apoptosis

• Cell types: Primary macrophages or differentiated THP-1 cells are preferred for inflammasome studies; epithelial or cancer cell lines are commonly used for apoptosis studies

How can I optimize Western blot detection of Cleaved-CASP7 (S199)?

For optimal Western blot results with Cleaved-CASP7 (S199) antibody:

• Sample preparation:

  • Include protease inhibitors in lysis buffer

  • Avoid freeze-thaw cycles of protein samples

  • For positive controls, treat Jurkat cells with etoposide (25 μM) for 5 hours

• Gel and transfer conditions:

  • Use 12-15% polyacrylamide gels for better resolution of the cleaved fragment (20 kDa)

  • Transfer at low voltage (30V) overnight at 4°C to ensure efficient transfer of small fragments

• Antibody conditions:

  • Start with 1:1000 dilution in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle agitation

  • Use HRP-conjugated secondary antibodies at 1:10000 dilution

• Signal development:

  • Use enhanced chemiluminescence (ECL) with appropriate exposure times

  • Consider signal enhancers if signal is weak

  • For difficult samples, ECL substrates with extended sensitivity may be required

• Troubleshooting high background:

  • Increase blocking time (1 hour to overnight)

  • Use 5% BSA instead of milk for blocking and antibody dilution

  • Include 0.2% Tween-20 in wash buffers

  • Ensure all incubations and washes are performed at recommended durations

Expected molecular weight of cleaved Caspase-7 is approximately 20 kDa, though the uncleaved proform appears at approximately 35 kDa .

How can Cleaved-CASP7 (S199) Antibody be used to investigate inflammasome-dependent activation?

The relationship between inflammasomes and Caspase-7 activation offers unique research opportunities:

• Experimental design for inflammasome activation:

  • Two-signal approach: Prime cells with LPS (200 ng/ml, 3h), then activate with ATP (5 mM, 0.5-1h)

  • Bacterial infection models: Infect macrophages with Salmonella (MOI 10-20) for inflammasome-dependent Caspase-7 activation

  • Controls should include cells from inflammasome component knockouts (NLRP3-/-, ASC-/-, Caspase-1-/-)

• Assessment methods:

  • Western blot for cleaved Caspase-7 alongside cleaved Caspase-1

  • Multiplex analysis of IL-1β, IL-18, and cleaved Caspase-7

  • Immunofluorescence co-staining for ASC specks and cleaved Caspase-7

  • Cell death assays (LDH release) to distinguish pyroptosis from apoptosis

• Important controls:

  • Pharmacological inhibition: Use Caspase-1 specific inhibitors to verify dependence

  • Genetic validation: Knockout or knockdown inflammasome components

  • Specificity verification: Monitor both Caspase-3 and Caspase-7 activation (Caspase-3 activation should be independent of inflammasome)

Research has demonstrated that during infection or inflammatory stimuli, Caspase-7 activation strictly depends on inflammasome components, whereas Caspase-3 activation proceeds independently, highlighting distinct activation mechanisms for these closely related proteases .

What are the considerations for using Cleaved-CASP7 (S199) Antibody in conjunction with inhibitors?

When using caspase inhibitors alongside Cleaved-CASP7 antibody detection:

• Inhibitor selection:

  • For Caspase-7 specific inhibition: Use selective Caspase-7 inhibitors

  • For upstream inhibition: Select inhibitors specific to initiator caspases (Caspase-8, -9, -10)

  • For inflammasome inhibition: Target either NLRP3 (MCC950) or Caspase-1 (VX-765)

• Experimental design considerations:

  • Include concentration gradients to establish dose-response relationships

  • Perform time-course studies to determine optimal inhibitor pre-treatment duration

  • Include positive controls (untreated but stimulated) and vehicle controls

  • Verify inhibitor efficacy using activity assays for targeted caspases

• Potential technical challenges:

  • Many commercial inhibitors have cross-reactivity with multiple caspases

  • Some inhibitors may exhibit cytotoxicity at higher concentrations

  • Inhibitors may affect antibody epitope accessibility in certain applications

  • Timing of inhibitor addition is critical (pre-treatment vs. co-treatment)

• Data interpretation:

  • Reduction in cleaved Caspase-7 signal with upstream inhibitors confirms pathway dependence

  • Persistence of cleaved Caspase-7 despite inhibitor treatment may indicate alternative activation pathways

  • Compare results between different detection methods (Western blot vs. immunofluorescence)

  • Consider combining inhibitor studies with genetic approaches for conclusive results

Experiments with inflammasome/Caspase-1 inhibitors have demonstrated the upstream requirement of this pathway for Caspase-7 activation during bacterial infection, while Caspase-3 activation remained unaffected , highlighting the utility of inhibitor studies in delineating selective activation pathways.