Cleaved-CASP9 (D330) Antibody

What is Cleaved-CASP9 (Asp330) and why is it significant in apoptosis research?

Cleaved-Caspase-9 (Asp330) represents the activated form of Caspase-9, which is cleaved at the aspartic acid residue at position 330 during apoptosis. This proteolytic processing is a critical event in the intrinsic apoptotic pathway. Caspase-9 functions as an initiator caspase that, once activated, can cleave and activate executioner caspases like Caspase-3 and Caspase-7. The detection of cleaved Caspase-9 serves as a reliable marker for the activation of the intrinsic apoptotic pathway, making it valuable in research focused on programmed cell death mechanisms, cancer therapies, and cellular stress responses . The antibody specifically recognizes the cleaved form at Asp330, enabling researchers to distinguish between inactive procaspase-9 and its activated form.

What are the key specifications of commercially available Cleaved-CASP9 (Asp330) antibodies?

Commercial antibodies targeting Cleaved-CASP9 (Asp330) have specific technical characteristics important for experimental design:

The antibodies are designed to be highly specific for the cleaved form of human Caspase-9, with endogenous sensitivity allowing detection of naturally occurring protein levels without overexpression systems .

How does Cleaved-CASP9 (Asp330) antibody differ from Cleaved-CASP9 (Asp315) antibody?

The key difference between these antibodies lies in the specific cleavage site they recognize:

• Cleaved-CASP9 (Asp330) antibody: Recognizes Caspase-9 cleaved at aspartic acid residue 330, resulting in a fragment of approximately 37 kDa .

• Cleaved-CASP9 (Asp315) antibody: Detects Caspase-9 cleaved at aspartic acid residue 315, producing a slightly smaller fragment of approximately 35 kDa .

These different cleavage sites represent distinct processing events in the apoptotic cascade. The Asp315 site is associated with processing by upstream caspases, while the Asp330 site may represent alternative processing mechanisms. Selecting the appropriate antibody depends on which specific cleavage product is of interest in your experimental system. Some studies may benefit from using both antibodies to comprehensively analyze different Caspase-9 processing events during apoptosis .

What are the optimal experimental conditions for detecting Cleaved-CASP9 (Asp330) by Western blotting?

For optimal Western blot detection of Cleaved-CASP9 (Asp330):

• Sample preparation: Lyse cells in a buffer containing protease inhibitors to prevent additional cleavage. Include phosphatase inhibitors if examining potential phosphorylation events that may influence caspase cleavage.

• Protein loading: Load 20-40 μg of total protein per lane. For apoptotic samples with low abundance of cleaved caspase, consider immunoprecipitation before Western blotting.

• Gel selection: Use 10-12% polyacrylamide gels to optimally resolve the 37 kDa cleaved fragment.

• Transfer considerations: Semi-dry or wet transfer protocols are suitable, with PVDF membranes providing optimal protein retention.

• Antibody dilution: Use 1:1000 dilution for primary antibody incubation (overnight at 4°C preferred) .

• Controls: Include positive controls (cells treated with apoptosis inducers like staurosporine) and negative controls (cells treated with pan-caspase inhibitors).

• Detection sensitivity: Use enhanced chemiluminescence (ECL) systems with longer exposure times if signal is weak, as cleaved products may be present at low abundance in early apoptosis.

The detection strategy should be optimized based on your specific experimental system and expected level of caspase activation .

How can I validate the specificity of the Cleaved-CASP9 (Asp330) antibody signal in my experimental system?

Validating antibody specificity for Cleaved-CASP9 (Asp330) requires multiple approaches:

• Positive controls: Include samples from cells treated with established apoptosis inducers (e.g., staurosporine, etoposide, UV radiation) to generate cleaved Caspase-9.

• Negative controls:

  • Pre-treatment with pan-caspase inhibitors (z-VAD-fmk) should abolish the signal

  • Caspase-9 knockout/knockdown cells should show no signal

  • Competing peptide blocking experiment using the immunizing peptide

• Parallel detection: Use multiple antibodies recognizing different epitopes of Caspase-9 to confirm similar patterns of activation.

• Size verification: Verify that the detected band corresponds to the expected molecular weight (37 kDa for the cleaved form) .

• Correlation with functional assays: Confirm that Cleaved-CASP9 detection correlates with downstream events (Caspase-3 activation, PARP cleavage).

• Time-course analysis: Signal should increase with the progression of apoptosis and follow expected kinetics.

This multi-faceted validation approach ensures that the detected signal truly represents the specific cleavage product of Caspase-9 at Asp330 rather than non-specific binding or similar-sized proteins .

What methods are available for quantifying Cleaved-CASP9 (Asp330) beyond traditional Western blotting?

Several methodologies provide complementary approaches to Western blotting:

• Cell-Based ELISA: Colorimetric or chemiluminescent ELISA kits allow quantitative detection of Cleaved-CASP9 (Asp330) directly in fixed cells in microplate format. This enables higher throughput screening and normalization to total cell number using secondary staining (e.g., crystal violet) .

• Flow Cytometry: Using permeabilization protocols and fluorescently-labeled secondary antibodies, cleaved Caspase-9 can be quantified at the single-cell level, allowing for population distribution analysis.

• Immunofluorescence Microscopy: Provides spatial information about Cleaved-CASP9 localization during apoptosis, with quantification possible through image analysis software.

• Proximity Ligation Assay (PLA): Enables detection of interactions between Cleaved-CASP9 and binding partners with higher sensitivity than co-immunoprecipitation.

• Mass Spectrometry: For absolute quantification of cleaved Caspase-9 products, targeted MS approaches using isotope-labeled internal standards can be employed.

• Luminex/Multiplex Assays: Allow simultaneous quantification of multiple apoptotic markers including Cleaved-CASP9.

Each method offers different advantages in terms of sensitivity, throughput, and the type of information provided. Selection should be based on your specific research questions and available equipment .

How can I use Cleaved-CASP9 (Asp330) antibody to investigate the kinetics of apoptosis activation?

To investigate apoptosis kinetics using Cleaved-CASP9 (Asp330) antibody:

• Time-course experimental design: Collect samples at multiple timepoints (e.g., 0, 1, 2, 4, 6, 12, 24 hours) after apoptotic stimulus.

• Quantitative Western blotting: Use internal loading controls and densitometry to quantify the relative abundance of cleaved Caspase-9 at each timepoint.

• Multiplex analysis: Simultaneously detect multiple apoptotic markers (Cleaved-CASP9, Cleaved-CASP3, Cleaved PARP, cytochrome c) to create a comprehensive activation profile.

• Single-cell analysis: Combine flow cytometry or immunofluorescence with Cleaved-CASP9 (Asp330) antibody to assess population heterogeneity in apoptosis timing.

• Correlation with functional readouts: Parallel assessment of mitochondrial membrane potential, phosphatidylserine externalization, and DNA fragmentation to correlate biochemical events with Caspase-9 cleavage.

• Mathematical modeling: Use quantitative data to develop kinetic models of the apoptotic cascade, establishing the temporal relationships between different events.

This approach enables determination of the precise timing of Caspase-9 activation relative to other apoptotic events, providing insights into the mechanisms and rate-limiting steps of apoptosis in your experimental system .

What are the considerations when using Cleaved-CASP9 (Asp330) antibody in co-immunoprecipitation studies?

When performing co-immunoprecipitation (co-IP) with Cleaved-CASP9 (Asp330) antibody:

• Lysis buffer composition: Use non-denaturing buffers that preserve protein-protein interactions while efficiently extracting Caspase-9 complexes. Include protease inhibitors to prevent additional cleavage events.

• Antibody concentration: The recommended 1:100 dilution for immunoprecipitation should be optimized for your specific system .

• Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Cross-linking considerations: In some cases, mild cross-linking before lysis may help preserve transient interactions of the activated caspase complex.

• Negative controls: Include IgG control immunoprecipitations and, if possible, samples from Caspase-9 knockout/knockdown cells.

• Detection strategy: When blotting co-immunoprecipitated samples, be aware that the heavy chain of the IP antibody (~50 kDa) may interfere with detection of proteins of similar size.

• Reciprocal confirmation: Confirm interactions by performing the co-IP in reverse, using antibodies against the suspected interacting partners.

• Alternative approaches: Consider proximity ligation assays or FRET-based approaches for detecting interactions in intact cells.

Remember that the cleaved form of Caspase-9 may have different interaction partners than the pro-form, and these interactions may be highly dynamic during the apoptotic process .

How can I differentiate between various cleavage forms of Caspase-9 in my experimental system?

Differentiating between various Caspase-9 cleavage products requires careful analytical approaches:

• Complementary antibodies: Use multiple antibodies recognizing distinct cleavage sites (Asp315, Asp330) and compare their detection patterns .

• Molecular weight analysis: Employ high-resolution SDS-PAGE (12-15% gels) to separate closely sized cleavage products:

  • Full-length procaspase-9: ~46-47 kDa

  • Large subunit (Asp315 cleavage): ~35 kDa

  • Large subunit (Asp330 cleavage): ~37 kDa

  • Small subunit (p10): ~10 kDa

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate products with similar molecular weights but different charges.

• Mass spectrometry: Use targeted MS approaches to identify specific cleavage sites based on peptide mass.

• Mutational analysis: Express Caspase-9 mutants with specific cleavage sites altered (D315A, D330A) as reference standards.

• In vitro cleavage assays: Generate reference standards by incubating recombinant procaspase-9 with purified caspases or apoptosome components.

• Time-course analysis: Different cleavage sites may be processed with distinct kinetics, allowing temporal discrimination.

This multi-faceted approach enables precise characterization of the Caspase-9 cleavage profile in response to different apoptotic stimuli .

What are potential causes of weak or absent Cleaved-CASP9 (Asp330) signal in apoptotic samples?

Several technical and biological factors may contribute to weak Cleaved-CASP9 (Asp330) signals:

• Technical limitations:

  • Insufficient sensitivity of detection system

  • Suboptimal antibody dilution (try 1:500 instead of 1:1000)

  • Inadequate protein loading (increase to 50-75 μg for low-abundance samples)

  • Protein degradation during sample preparation (enhance protease inhibition)

  • Inefficient protein transfer (optimize transfer conditions)

  • Excessive washing (reduce stringency)

• Biological considerations:

  • Timing: Caspase-9 activation may be transient or occur at a different timepoint than examined

  • Stimulus-specific activation: Not all apoptotic stimuli strongly activate the intrinsic pathway

  • Cell type-specific responses: Certain cell lines may preferentially utilize alternative pathways

  • Inhibitory mechanisms: Endogenous inhibitors (IAPs, XIAP) may prevent Caspase-9 cleavage

  • Rapid degradation: Cleaved products may be quickly degraded in some systems

• Experimental approach adjustments:

  • Perform immunoprecipitation before Western blotting to concentrate the target protein

  • Use positive control samples from cells treated with strong intrinsic pathway activators

  • Consider alternative detection methods (ELISA, immunofluorescence)

  • Verify apoptosis is occurring using parallel markers (Annexin V, PARP cleavage)

When troubleshooting, systematically address both technical and biological variables before concluding that Caspase-9 activation is absent in your system .

How can I distinguish between specific Cleaved-CASP9 (Asp330) signal and non-specific antibody binding?

Distinguishing specific from non-specific signals requires rigorous controls:

• Positive and negative controls:

  • Include samples known to express (apoptotic cells) or lack (non-apoptotic cells) the target

  • Use Caspase-9 knockout/knockdown cells as definitive negative controls

  • Include pan-caspase inhibitor-treated samples as functional negative controls

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide before Western blotting

  • Specific bands should disappear while non-specific bands persist

• Molecular weight verification:

  • Cleaved-CASP9 (Asp330) should appear at approximately 37 kDa

  • Multiple, non-predicted bands suggest non-specific binding

• Antibody validation approaches:

  • Test multiple antibodies against different epitopes of Caspase-9

  • Compare monoclonal and polyclonal antibody staining patterns

  • Validate with orthogonal methods (mass spectrometry, activity assays)

• Stringency optimization:

  • Increase blocking agent concentration (5% BSA or milk)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Increase salt concentration in wash buffers to reduce non-specific ionic interactions

• Sample preparation considerations:

  • Include reducing agents in sample buffer to minimize non-specific disulfide bonds

  • Optimize lysis conditions to reduce sample complexity and background

These approaches collectively provide confidence in the specificity of detected Cleaved-CASP9 signals .

What experimental artifacts might affect Cleaved-CASP9 (Asp330) detection and how can they be mitigated?

Several experimental artifacts can influence Cleaved-CASP9 detection:

• Post-lysis activation artifacts:

  • Problem: Caspase activation occurring after cell lysis rather than in vivo

  • Solution: Include caspase inhibitors in lysis buffer; keep samples cold; process rapidly

• Sample degradation:

  • Problem: Proteolytic degradation producing fragments similar to specific cleavage products

  • Solution: Use fresh protease inhibitor cocktails; avoid freeze-thaw cycles; prepare samples immediately before analysis

• Antibody cross-reactivity:

  • Problem: Recognition of similar epitopes in related proteins (other caspases)

  • Solution: Verify specificity with knockout controls; use monoclonal antibodies; perform peptide competition assays

• Incomplete transfer artifacts:

  • Problem: Inconsistent transfer efficiency across the gel

  • Solution: Use transfer control stains; optimize transfer conditions for target protein size

• Loading control inconsistencies:

  • Problem: Traditional loading controls may change during apoptosis

  • Solution: Use total protein normalization (Ponceau S, REVERT); validate multiple loading controls

• Cell density artifacts:

  • Problem: Variations in apoptosis sensitivity due to cell confluence differences

  • Solution: Standardize plating density; use cell-based ELISAs with normalization to cell number

• Treatment-induced modifications:

  • Problem: Post-translational modifications affecting antibody recognition

  • Solution: Test multiple antibodies recognizing different epitopes; validate with activity assays

• Detergent incompatibility:

  • Problem: Certain detergents may affect epitope accessibility

  • Solution: Test alternative lysis buffers; optimize detergent concentration

Addressing these potential artifacts ensures that observed changes in Cleaved-CASP9 truly reflect biological processes rather than technical variables .

How can Cleaved-CASP9 (Asp330) antibody be used to investigate non-apoptotic caspase functions?

Beyond apoptosis, Cleaved-CASP9 (Asp330) antibody can investigate emerging non-canonical functions:

• Cellular differentiation studies:

  • Monitor sub-lethal Caspase-9 activation during differentiation processes

  • Correlate activation patterns with stage-specific differentiation markers

  • Use immunofluorescence to track subcellular localization during differentiation

• Inflammasome research:

  • Investigate potential crosstalk between inflammasome components and Caspase-9

  • Examine Caspase-9 activation in response to DAMPs and PAMPs

  • Compare activation kinetics with inflammatory caspases

• Neurodegenerative disease models:

  • Detect low-level, chronic Caspase-9 activation in neurons

  • Correlate with synaptic dysfunction before overt cell death

  • Use proximity ligation assays to identify novel interaction partners in neurons

• Cell cycle regulation:

  • Monitor Caspase-9 cleavage during specific cell cycle phases

  • Combine with cyclin markers to establish precise temporal relationships

  • Investigate potential substrates in cell cycle regulation

• Autophagy interactions:

  • Examine Caspase-9 activity during autophagic processes

  • Investigate potential cleavage of autophagy-related proteins

  • Study cross-regulation between apoptotic and autophagic pathways

• Proteomic approaches:

  • Immunoprecipitate Cleaved-CASP9 complexes for mass spectrometry analysis

  • Identify novel non-apoptotic binding partners

  • Discover new potential substrates in various cellular processes

These applications expand our understanding of Caspase-9 beyond its canonical role in apoptosis, potentially revealing new therapeutic targets for diseases involving dysregulated cellular processes .

What considerations are important when using Cleaved-CASP9 (Asp330) antibody in tissue samples versus cell culture?

Working with tissue samples introduces several important considerations compared to cell culture:

• Fixation effects:

  • Formalin fixation may mask epitopes recognized by the antibody

  • Optimize antigen retrieval protocols (citrate buffer, EDTA, enzymatic)

  • Consider testing multiple antibody clones for compatibility with fixed tissues

• Tissue heterogeneity:

  • Cell-specific expression levels may dilute signals in whole tissue lysates

  • Consider laser capture microdissection to isolate specific cell populations

  • Use immunohistochemistry to identify cell-specific activation patterns

• Post-mortem changes:

  • Caspases may activate during post-mortem interval, creating artifacts

  • Analyze time-course of post-mortem changes to establish baselines

  • Include appropriate time-matched controls

• Perfusion considerations:

  • Blood contamination may affect interpretation of results

  • Consider perfusion-fixed tissues for cleaner backgrounds

  • Include non-perfused controls to understand impact

• Technical adaptations:

  • Tissue homogenization must be optimized to release caspases efficiently

  • Higher detergent concentrations may be necessary for complete extraction

  • Increased antibody concentrations may be required (1:500 instead of 1:1000)

• Validation approaches:

  • Correlate Western blot findings with immunohistochemistry

  • Use multiple antibodies recognizing different epitopes

  • Include tissue from Caspase-9 knockout models as controls

• Spatial considerations:

  • Caspase activation may be localized to specific tissue regions

  • Use immunofluorescence to preserve spatial information

  • Consider tissue microarrays for high-throughput screening

These adaptations ensure reliable detection of Cleaved-CASP9 (Asp330) in complex tissue environments while minimizing artifacts .

How does phosphorylation status affect Caspase-9 cleavage and detection with Cleaved-CASP9 (Asp330) antibody?

Phosphorylation significantly impacts Caspase-9 biology and detection:

• Known phosphorylation sites:

  • Multiple residues including Ser99, Thr125, Ser144, Ser183, Tyr153, and others have been identified

  • Different kinases target specific sites (Akt, ERK, CDKs, c-Abl)

• Functional consequences:

  • Inhibitory phosphorylation: Many sites (especially Ser196 by Akt) inhibit Caspase-9 activation

  • Structural changes: Phosphorylation can alter procaspase-9 conformation

  • Apoptosome interaction: Some modifications affect APAF-1 binding

  • Cleavage accessibility: Phosphorylation near cleavage sites may affect protease accessibility

• Detection implications:

  • Phosphorylation near the Asp330 site may affect antibody recognition

  • Sample preparation should preserve phosphorylation status (phosphatase inhibitors)

  • Dephosphorylation may increase susceptibility to cleavage in lysates

• Experimental approaches:

  • Compare phosphatase-treated and untreated samples

  • Use phospho-specific antibodies in parallel with Cleaved-CASP9 detection

  • Express phosphomimetic and phospho-deficient mutants as references

  • Employ Phos-tag gels to separate phosphorylated forms before immunoblotting

• Kinase inhibitor studies:

  • Treat cells with specific kinase inhibitors before apoptotic stimulus

  • Monitor changes in Cleaved-CASP9 detection efficiency

  • Correlate with changes in apoptotic sensitivity

Understanding the interplay between phosphorylation and cleavage provides insight into the regulatory mechanisms controlling the apoptotic threshold in different cellular contexts .