Cleaved-CASP9 (D315) Antibody

What is Cleaved-CASP9 (D315) Antibody and what epitope does it recognize?

Cleaved-CASP9 (D315) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of the large fragment (p35) of activated Caspase-9 protein resulting from cleavage adjacent to aspartic acid 315 . This antibody recognizes the neoepitope that is exposed only after the cleavage event, making it a valuable tool for detecting activated caspase-9 in apoptotic cells. The antibody does not recognize full-length, inactive procaspase-9, ensuring specificity for the activated form .

What is the biological significance of Caspase-9 cleavage at D315?

Caspase-9 is a critical initiator caspase in the intrinsic apoptotic pathway. When cells receive apoptotic signals through the mitochondrial pathway, cytochrome c is released, which then binds to Apaf-1 to form the apoptosome. This complex recruits and activates procaspase-9 through dimerization and subsequent cleavage at D315 . This cleavage generates an active form of caspase-9 (p35/p10) that activates executioner caspases like caspase-3 and caspase-7, leading to cellular dismantling during apoptosis .

How does D315 cleavage differ from other caspase-9 processing events?

Caspase-9 can be cleaved at multiple sites, with D315 and D330 being the most significant:

• D315 cleavage: The primary autocatalytic cleavage site during apoptosome-mediated activation, generating a 35 kDa fragment that is recognized by Cleaved-CASP9 (D315) Antibody .

• D330 cleavage: A secondary site processed by active caspase-3, generating an alternate neoepitope .

Both D315 and D330 cleaved forms are fully active proteases, but they are differentially regulated: the D315 form is selectively inhibited by the Bir3 domain of XIAP (X-linked Inhibitor of Apoptosis Protein), while the D330 form is less susceptible to this inhibition . Therefore, detection of these different cleaved forms using site-specific antibodies provides insights into the progression and regulation of apoptotic pathways.

What applications has Cleaved-CASP9 (D315) Antibody been validated for?

The Cleaved-CASP9 (D315) Antibody has been validated for multiple research applications:

These applications enable comprehensive analysis of caspase-9 activation in various experimental systems .

What is the recommended protocol for Western blotting with Cleaved-CASP9 (D315) Antibody?

For optimal Western blot results with Cleaved-CASP9 (D315) Antibody:

• Prepare protein lysates from cells undergoing apoptosis (treatment with staurosporine, etoposide, or other apoptotic inducers as positive controls)

• Separate proteins by SDS-PAGE (10-12% gel recommended)

• Transfer proteins to PVDF or nitrocellulose membrane

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

• Incubate with Cleaved-CASP9 (D315) Antibody at 1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Wash 3-5 times with TBST

• Detect signal using ECL substrate

• Expected band size: 35-37 kDa corresponding to the large subunit of cleaved caspase-9

How should samples be prepared to maximize detection of cleaved Caspase-9?

To maximize detection of cleaved caspase-9:

• Timing is critical: Harvest cells at multiple time points after apoptotic stimulation to capture the optimal window of caspase-9 activation

• Use protease inhibitors: Include complete protease inhibitor cocktail in lysis buffer

• Rapid sample processing: Minimize time between cell harvesting and lysis to prevent artificial activation or degradation

• Sample storage: Store samples at -80°C with 50% glycerol to prevent degradation

• Positive controls: Include samples treated with known apoptotic inducers like staurosporine

• Phosphatase inhibitors: Include these in lysis buffer to preserve phosphorylation status that may affect caspase-9 cleavage

What are the storage and handling recommendations for Cleaved-CASP9 (D315) Antibody?

For optimal antibody performance:

• Storage temperature: -20°C for up to 1 year

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Formulation: The antibody is supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Working dilutions should be prepared fresh and used within 24 hours

• For long-term storage, keep antibody in its original container and avoid exposure to light

How can Cleaved-CASP9 (D315) Antibody be used to differentiate between intrinsic and extrinsic apoptotic pathways?

The intrinsic and extrinsic apoptotic pathways can be differentiated using Cleaved-CASP9 (D315) Antibody in combination with other apoptotic markers:

• Experimental design approach:

  • Treat cells with pathway-specific inducers:

    • Intrinsic pathway: DNA damaging agents (etoposide, cisplatin)

    • Extrinsic pathway: Death receptor ligands (TNF-α, FasL, TRAIL)

  • Perform time-course analysis to capture the sequence of events

  • Use a panel of antibodies for Western blotting or immunofluorescence:

    • Cleaved-CASP9 (D315): Marker for intrinsic pathway activation

    • Cleaved-CASP8: Marker for extrinsic pathway activation

    • Cytochrome c (cytosolic fraction): Upstream event in intrinsic pathway

    • Cleaved-CASP3: Downstream event in both pathways

• Data interpretation:

  • Intrinsic pathway predominance: Early and strong cleaved-CASP9 signal preceding cleaved-CASP3

  • Extrinsic pathway predominance: Strong cleaved-CASP8 signal with delayed or weak cleaved-CASP9

  • Cross-talk between pathways: Both cleaved-CASP8 and cleaved-CASP9 signals with variable timing

How can post-translational modifications of Caspase-9 be studied in relation to its cleavage at D315?

Caspase-9 is regulated by multiple post-translational modifications (PTMs) that affect its cleavage and activity. To study these:

• Experimental approaches:

  • Phosphorylation analysis:

    • Treat cells with kinase activators/inhibitors

    • Immunoprecipitate caspase-9 and probe with phospho-specific antibodies (pThr125, pTyr153)

    • Use phosphomimetic or phospho-dead mutants

  • Ubiquitination analysis:

    • Treat cells with proteasome inhibitors

    • Co-immunoprecipitation with ubiquitin antibodies

  • ADP-ribosylation detection:

    • Use specific ADP-ribosylation inhibitors

    • Perform mass spectrometry analysis to identify modification sites

• Correlation with D315 cleavage:

  • Perform parallel detection with Cleaved-CASP9 (D315) Antibody

  • Analyze time-course correlation between PTMs and cleavage events

  • Use site-directed mutagenesis to create caspase-9 variants resistant to specific PTMs and assess impact on D315 cleavage

What is the relationship between D315 cleavage of Caspase-9 and XIAP inhibition?

The relationship between D315 cleavage and XIAP inhibition is significant for understanding apoptotic regulation:

• XIAP selectively inhibits the D315-cleaved form of caspase-9 through its BIR3 domain, but has less effect on the D330-cleaved form .

• Experimental approaches to study this relationship:

  • Co-immunoprecipitation of cleaved caspase-9 with XIAP

  • Comparison of caspase-9 activity in the presence/absence of XIAP

  • Use of BIR3 domain mutants to disrupt the interaction

  • Simultaneous detection of both D315 and D330 cleaved forms to assess inhibition patterns

• Functional implications:

  • The D330 cleavage may represent a mechanism to overcome XIAP-mediated inhibition

  • This differential inhibition creates a regulatory checkpoint in the apoptotic cascade

  • XIAP levels may determine whether D315-cleaved caspase-9 can effectively activate downstream caspases

How can Cleaved-CASP9 (D315) Antibody be used in multiplexed detection systems?

Multiplexed detection with Cleaved-CASP9 (D315) Antibody allows comprehensive analysis of apoptotic signaling:

• Immunofluorescence multiplexing:

  • Co-stain with markers for:

    • Other caspases (cleaved caspase-3, cleaved caspase-8)

    • Mitochondrial proteins (cytochrome c, Bax)

    • DNA fragmentation (TUNEL assay)

    • Cell type-specific markers to identify vulnerable populations

• Flow cytometry applications:

  • Combined with membrane integrity dyes (PI, 7-AAD)

  • Multi-parameter analysis with Annexin V and other apoptotic markers

  • Cell cycle analysis to correlate with specific phases

• Protein array technologies:

  • Reverse phase protein arrays for high-throughput screening

  • Proximity ligation assays to detect protein-protein interactions involving cleaved caspase-9

What are common issues when using Cleaved-CASP9 (D315) Antibody and how can they be resolved?

How can researchers distinguish between caspase inhibition versus lack of activation?

Distinguishing between inhibition of active caspase-9 and absence of caspase-9 activation:

• Experimental approaches:

  • Parallel detection of:

    • Procaspase-9 levels: Decreased in activation, unchanged in inhibition

    • Cleaved caspase-9: Absent in both scenarios

    • Upstream events (cytochrome c release): Present in inhibition, absent in lack of activation

    • Downstream events (caspase-3 activation): Absent in both scenarios

• Specific scenarios to test:

  • Chemical inhibition:

    • Treat cells with pan-caspase inhibitors (z-VAD-fmk) or caspase-9 specific inhibitors

    • Should see upstream events but no cleaved caspase-9 or downstream activation

  • XIAP-mediated inhibition:

    • XIAP overexpression or BIR3 domain expression

    • May see partial caspase-9 cleavage but inhibited activity

• Enzyme activity assays:

  • Use fluorogenic substrates specific for caspase-9 to directly measure enzymatic activity

  • Compare with Western blot detection of cleaved forms to distinguish between presence and activity

How should researchers interpret contradictory results between D315 cleavage and other apoptotic markers?

Discrepancies between cleaved caspase-9 and other apoptotic markers may reveal important biological insights:

• Possible biological explanations:

  • Differential timing of events:

    • Caspase-9 activation may precede or follow other markers depending on context

    • Time-course experiments can resolve temporal discrepancies

  • Pathway-specific activation:

    • Caspase-9 specific to intrinsic pathway; may be absent in pure extrinsic activation

    • Caspase-independent cell death processes may show other markers without caspase-9

  • Feedback and amplification loops:

    • Executioner caspases can cleave initiator caspases creating complex activation patterns

    • Inhibitory mechanisms may selectively block certain caspases

• Technical considerations:

  • Antibody sensitivity differences:

    • Compare detection limits of different antibodies

    • Some antibodies may require signal amplification systems

  • Epitope availability issues:

    • Protein-protein interactions may mask epitopes

    • Different fixation methods may affect epitope accessibility

What are best practices for quantifying Cleaved-CASP9 (D315) signal in research applications?

For rigorous quantification of cleaved caspase-9 signals:

• Western blot quantification:

  • Densitometry analysis using appropriate software

  • Normalization approaches:

    • Normalize to total caspase-9 (ratio of cleaved to total)

    • Normalize to housekeeping proteins (β-actin, GAPDH) for loading control

  • Present data as fold-change relative to control conditions

  • Include biological replicates (n≥3) for statistical analysis

• Immunofluorescence quantification:

  • Single-cell analysis approaches:

    • Count percentage of cells positive for cleaved caspase-9

    • Measure signal intensity per cell (integrated density)

    • Analyze subcellular distribution patterns

  • Use automated image analysis platforms

  • Assess co-localization with other markers

• Statistical considerations:

  • Appropriate statistical tests based on experimental design

  • Report p-values with appropriate significance thresholds

  • Consider biological vs. technical replicates in experimental design