Cleaved-CASP7 (D198) Antibody

What is Cleaved-CASP7 (D198) Antibody and what does it specifically detect?

Cleaved-CASP7 (D198) Antibody is a polyclonal antibody that specifically recognizes the cleaved form of Caspase-7 at the Aspartic Acid-198 position. It detects endogenous levels of the activated Caspase-7 p20 protein fragment resulting from proteolytic cleavage adjacent to D198, a critical event in the apoptotic cascade . This antibody binds to the amino acid region 149-198 in human Caspase-7 . The specificity for the cleaved form makes it particularly valuable for studying apoptotic processes where Caspase-7 activation occurs.

What cellular processes involve Cleaved-CASP7 and why is its detection important?

Cleaved-CASP7 is a crucial marker in programmed cell death pathways. Caspase-7 undergoes cleavage and activation by initiator caspases (CASP8, CASP9, CASP10), leading to the execution phase of apoptosis . Additionally, cleavage and maturation by granzyme B (GZMB) regulates granzyme-mediated programmed cell death, while CASP1 activates it in response to bacterial infection . The detection of cleaved Caspase-7 provides researchers with direct evidence of apoptotic pathway activation, making it valuable for studying cellular responses to various stimuli, disease mechanisms, and potential therapeutic interventions targeting programmed cell death.

What experimental applications are suitable for Cleaved-CASP7 (D198) Antibody?

Cleaved-CASP7 (D198) Antibody is applicable in several research techniques:

• Western Blot (recommended dilution 1:500-1:2000)

• ELISA (recommended dilution 1:40000)

• Immunohistochemistry (IHC)

• Immunoprecipitation (IP)

This versatility enables researchers to detect cleaved Caspase-7 in various experimental setups, including cell lysates, tissue samples, and potentially in situ detection in fixed specimens, providing multiple approaches to investigate apoptotic processes in different research contexts.

How should I optimize Western blot protocols for detecting Cleaved-CASP7?

For optimal Western blot results with Cleaved-CASP7 (D198) Antibody:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent further degradation of proteins after collection.

• Loading control: Include appropriate loading controls, especially when comparing treated versus untreated samples.

• Dilution optimization: Start with a 1:1000 dilution and adjust based on signal strength.

• Positive control: Include a positive control such as Jurkat cells treated with apoptosis-inducing agents like etoposide (25μM for 24h) , which has been demonstrated to produce detectable levels of cleaved Caspase-7.

• Blocking verification: Consider running a parallel experiment with the synthesized peptide blocking to confirm specificity, as shown in validation studies .

The molecular weight of the cleaved Caspase-7 p20 fragment is approximately 20 kDa , which should be your target band for identification.

What are the critical factors for successful cell-based ELISA using Cleaved-CASP7 (D198) Antibody?

When performing cell-based ELISA with Cleaved-CASP7 (D198) Antibody:

• Cell type selection: The antibody works effectively with human and mouse adherent cell lines .

• Cell density optimization: Establish optimal seeding density to ensure consistent cell numbers across wells.

• Fixation protocol: Proper fixation is critical to preserve the cleaved epitope while maintaining cellular architecture.

• Apoptosis induction: Consider time-course experiments to capture the optimal window for Caspase-7 cleavage following stimulus application.

• Normalization strategy: Include parallel wells for total protein or housekeeping protein detection to normalize Cleaved-CASP7 signals.

• Signal-to-background ratio: Optimize blocking conditions and antibody concentrations to minimize background while maximizing specific signal.

This approach allows for quantitative assessment of apoptosis in adherent cells without the need for lysate preparation .

How can I validate the specificity of Cleaved-CASP7 (D198) signal in my experimental system?

Validation strategies for confirming Cleaved-CASP7 (D198) Antibody specificity include:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites before application to samples .

• Positive and negative controls: Include known apoptotic (e.g., etoposide-treated Jurkat cells) and non-apoptotic control samples.

• Caspase inhibitor experiments: Pre-treat cells with pan-caspase or Caspase-7-specific inhibitors to prevent cleavage.

• Genetic validation: Use CASP7 knockout or knockdown samples as negative controls.

• Parallel detection: Employ alternative methods or antibodies recognizing different epitopes of cleaved Caspase-7 to confirm results.

• Time-course analysis: Monitor the appearance of the cleaved form following apoptotic stimulus application to confirm the expected kinetics.

How can Cleaved-CASP7 (D198) Antibody be used to distinguish between different modes of cell death?

Cleaved-CASP7 (D198) Antibody can help differentiate between apoptotic and non-apoptotic cell death through:

• Comparative pathway analysis: Combine with markers for other cell death pathways (necroptosis, pyroptosis, ferroptosis) to create a comprehensive profile.

• Temporal analysis: Monitor the sequence of caspase activation (initiator vs. executioner) to distinguish intrinsic versus extrinsic apoptosis.

• Stimulus-specific responses: Compare CASP7 cleavage patterns between death receptor activation (e.g., TNF-α, FasL) and mitochondrial pathway inducers (e.g., staurosporine).

• Co-localization studies: Combine with subcellular markers to determine the compartmentalization of cleaved Caspase-7 during different death processes.

• Quantitative assessment: Measure the ratio of cleaved to uncleaved Caspase-7 to evaluate the completeness of the apoptotic response.

This approach provides insight into the specific cell death mechanisms activated in response to various experimental conditions or disease states.

What are the considerations for multiplexing Cleaved-CASP7 detection with other apoptotic markers?

For multiplexed detection strategies:

• Antibody compatibility: When combining antibodies, ensure they are raised in different host species or use directly conjugated primary antibodies to avoid cross-reactivity.

• Sequential detection approach: Consider the optimal sequence for multiple detections:

  • Start with the lowest abundance target (often cleaved caspases)

  • Strip and reprobe, or use spectrally distinct fluorophores

• Relevant marker combinations:

  • Initiator caspases (cleaved CASP8, CASP9) to determine apoptotic pathway origin

  • Other executioner caspases (cleaved CASP3) to assess pathway completeness

  • Substrate cleavage (PARP) to confirm functional consequences

• Controls for each marker: Include single-marker controls to verify that multiplexing doesn't compromise individual signal detection.

• Quantitative considerations: Account for potential signal overlap or interference in quantitative analyses.

This multiplexed approach provides a comprehensive view of the apoptotic cascade activation in experimental systems.

How can Cleaved-CASP7 (D198) Antibody be utilized in drug discovery research targeting apoptotic pathways?

In drug discovery applications:

• High-throughput screening: Adapt cell-based ELISA formats for plate-based screening of compounds that modulate apoptosis.

• Dose-response studies: Quantify Cleaved-CASP7 levels across concentration ranges to establish EC50/IC50 values for pro- or anti-apoptotic compounds.

• Temporal dynamics: Determine the kinetics of Caspase-7 activation in response to drug treatments to optimize dosing schedules.

• Combination therapy assessment: Evaluate synergistic effects of drug combinations on apoptotic pathway activation.

• Target validation: Use siRNA/CRISPR approaches alongside antibody detection to confirm the role of specific proteins in drug-induced apoptosis.

• Resistance mechanisms: Investigate changes in Caspase-7 cleavage patterns in drug-resistant versus sensitive cell populations.

This approach facilitates the development of therapeutics targeting programmed cell death pathways in cancer, neurodegenerative diseases, and other disorders.

What are the common technical challenges when using Cleaved-CASP7 (D198) Antibody and how can they be addressed?

Addressing these challenges systematically will improve detection reliability and experimental reproducibility.

How should I interpret differences in Cleaved-CASP7 expression between experimental conditions?

When interpreting variations in Cleaved-CASP7 detection:

What controls should be included when designing experiments using Cleaved-CASP7 (D198) Antibody?

A robust experimental design should include:

• Positive controls:

  • Jurkat cells treated with etoposide (25μM, 24h)

  • Cell lines with known apoptotic responses to standard inducers

  • Recombinant cleaved Caspase-7 protein (for Western blot standardization)

• Negative controls:

  • Untreated cells

  • Cells pre-treated with caspase inhibitors

  • Samples where antibody is pre-absorbed with immunizing peptide

• Technical controls:

  • Loading controls (β-actin, GAPDH) for Western blots

  • Isotype control antibodies for immunostaining applications

  • No-primary-antibody controls to assess secondary antibody specificity

• Validation controls:

  • Detection with alternative antibodies against the same target

  • Alternative methods to detect apoptosis (Annexin V, TUNEL)

  • CASP7 knockout/knockdown samples where available

Incorporating these controls enables confident interpretation of experimental results and identification of potential technical artifacts.

How can Cleaved-CASP7 (D198) Antibody be applied in single-cell analysis of heterogeneous populations?

For single-cell applications:

• Flow cytometry optimization: Adapt protocols for intracellular staining of Cleaved-CASP7 to enable high-throughput analysis of individual cells.

• Imaging cytometry: Combine with other markers to phenotype subpopulations exhibiting differential apoptotic responses.

• Single-cell sorting: Use Cleaved-CASP7 detection to isolate and further characterize apoptotic versus non-apoptotic cells from heterogeneous samples.

• Spatial considerations: In tissue sections, analyze the distribution of Cleaved-CASP7-positive cells relative to microenvironmental features.

• Resistance identification: Identify rare non-responsive cells in otherwise apoptosis-sensitive populations.

• Temporal heterogeneity: Track the asynchronous nature of apoptosis activation across a population over time.

These approaches reveal cell-to-cell variability in apoptotic responses that might be masked in bulk analyses, providing insight into resistance mechanisms and cellular decision-making processes.

What considerations are important when using Cleaved-CASP7 (D198) Antibody in tissue microenvironment studies?

For tissue-based investigations:

• Fixation optimization: Different fixatives can affect epitope accessibility; compare paraformaldehyde, formalin, and alcohol-based fixatives.

• Antigen retrieval: Determine optimal retrieval methods (heat-induced versus enzymatic) for the specific tissue type.

• Context-dependent interpretation: Consider that Cleaved-CASP7 detection in tissues represents a snapshot of an ongoing process.

• Spatial distribution analysis: Examine patterns of apoptotic cells relative to:

  • Tissue architecture (tumor margins, invasive fronts)

  • Vascular structures

  • Immune infiltrates

• Multi-marker integration: Combine with markers of tissue stress, hypoxia, or immune activity for comprehensive characterization.

• Quantitative approaches: Develop robust image analysis algorithms to quantify Cleaved-CASP7-positive cells across tissue regions.

This contextual approach reveals how microenvironmental factors influence apoptotic responses in complex tissues, providing insights beyond what cell culture studies can offer.

How might Cleaved-CASP7 detection contribute to understanding non-apoptotic functions of caspases?

For investigating non-canonical caspase functions:

• Sub-lethal activation: Detect low-level Caspase-7 cleavage in non-dying cells to identify potential signaling roles.

• Subcellular localization: Determine if cleaved Caspase-7 localizes to specific compartments during non-apoptotic processes.

• Stimulus specificity: Compare patterns of Caspase-7 cleavage between apoptotic versus non-apoptotic stimuli (e.g., differentiation signals, inflammatory triggers).

• Substrate analysis: Correlate Cleaved-CASP7 detection with non-apoptotic substrate cleavage events.

• Temporal dynamics: Examine whether transient versus sustained Caspase-7 activation correlates with different cellular outcomes.

• Experimental manipulation: Use optogenetic or chemical biology approaches to activate Caspase-7 without triggering full apoptosis.

This research direction could reveal novel roles for Caspase-7 in cellular processes beyond cell death, an emerging area of interest in the field.

What emerging technologies might enhance the utility of Cleaved-CASP7 (D198) Antibody in research?

Innovative approaches that could extend antibody applications include:

• Proximity ligation assays: Detect interactions between cleaved Caspase-7 and potential binding partners or substrates in situ.

• Live-cell compatible intrabodies: Develop cell-permeable derivatives to monitor Caspase-7 activation in real-time.

• Super-resolution microscopy: Apply advanced imaging to resolve the subcellular distribution of cleaved Caspase-7 at nanoscale resolution.

• Mass cytometry (CyTOF): Incorporate metal-tagged antibodies against Cleaved-CASP7 into high-dimensional single-cell analyses.

• Spatial transcriptomics integration: Correlate Cleaved-CASP7 protein detection with gene expression profiles in the same tissue regions.

• Microfluidic applications: Adapt detection for continuous monitoring of apoptosis in organ-on-chip models.

These technological advances could provide unprecedented insights into the spatial, temporal, and functional aspects of Caspase-7 activation in complex biological systems.

How can researchers leverage Cleaved-CASP7 detection in translational research applications?

Translational applications include:

• Biomarker development: Evaluate Cleaved-CASP7 as a potential prognostic or predictive biomarker in patient samples.

• Therapeutic response assessment: Monitor changes in Cleaved-CASP7 levels as pharmacodynamic markers of response to apoptosis-inducing therapies.

• Patient stratification: Determine if baseline or inducible Caspase-7 activation correlates with clinical outcomes or treatment responses.

• Ex vivo drug sensitivity testing: Use rapid Cleaved-CASP7 detection to assess patient-derived samples' responses to potential therapeutics.

• Companion diagnostics: Develop standardized assays to identify patients likely to respond to apoptosis-modulating therapies.

• Monitoring minimal residual disease: Detect rare apoptosis-resistant cells in post-treatment samples.

These approaches could bridge fundamental research on apoptotic mechanisms with clinical applications, potentially improving treatment selection and monitoring in diseases where dysregulated apoptosis plays a role.