Cleaved-CASP3 (D175) Antibody

What is the cleaved-CASP3 (D175) antibody and how does it differ from antibodies targeting total Caspase-3?

The cleaved-CASP3 (D175) antibody is a polyclonal antibody typically raised in rabbit against a peptide in the large subunit of human Caspase-3, specifically amino-terminal to Asp175. Unlike antibodies detecting total Caspase-3, this antibody only recognizes the cleaved form that appears after proteolytic separation between Asp175 and Ser176, which exposes the epitope. This makes it a specific marker for activated Caspase-3 in cells undergoing apoptosis . The antibody does not detect the unprocessed, inactive form of Caspase-3, making it valuable for specifically identifying cells committed to the apoptotic pathway .

What is the specific epitope recognized by cleaved-CASP3 (D175) antibody?

The cleaved-CASP3 (D175) antibody typically recognizes the tripeptide ETD (Glu-Thr-Asp) sequence that becomes exposed after proteolytic cleavage. This sequence is part of the larger epitope structure CRGTELDCGIETD in the large subunit of Caspase-3 . The specificity for this region allows the antibody to detect only the activated form of Caspase-3, making it a reliable marker for cells undergoing apoptosis .

What applications is the cleaved-CASP3 (D175) antibody validated for?

The cleaved-CASP3 (D175) antibody has been validated for several experimental applications including:

• Western Blot (WB) at dilutions of 1:500-2000

• Immunohistochemistry on paraffin-embedded sections (IHC-p) at dilutions of 1:50-300

• Immunofluorescence (IF) at dilutions of 1:50-300

It has been verified with multiple sample types including human cell lines (e.g., HeLa), mouse tissues (colon, kidney), rat tissues (heart, lung, brain, liver), and human cancer tissues (stomach cancer) .

How should I design a Western blot experiment to detect cleaved Caspase-3?

For optimal detection of cleaved Caspase-3 by Western blot:

• Sample preparation: Induce apoptosis in your cells using appropriate treatment (e.g., staurosporine at 1 μM is commonly used)

• Time course: Include multiple time points (2-6 hours is often optimal) to capture the window of Caspase-3 activation

• Controls: Include both positive controls (apoptosis-induced cells) and negative controls (untreated cells)

• Gel percentage: Use 12-15% gels for better resolution of the cleaved fragment

• Expected bands: Look for bands at approximately 17-20 kDa, which represent the cleaved large subunit

Note that while the calculated molecular weight is 32 kDa for full-length Caspase-3, the cleaved product observed in Western blots typically appears at approximately 17-20 kDa .

What are the critical methodological considerations for immunofluorescence experiments with cleaved-CASP3 (D175) antibody?

For successful immunofluorescence experiments:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilization: Use 0.1-0.3% Triton X-100 for 10 minutes

• Blocking: Block with 5% normal serum (from the species of secondary antibody) for 1 hour

• Primary antibody: Use cleaved-CASP3 (D175) at dilution 1:50-300

• Incubation time: Incubate primary antibody for at least 3 hours at room temperature or overnight at 4°C

• Secondary antibody: Use appropriate fluorochrome-conjugated secondary antibody (e.g., anti-rabbit IgG)

• Counterstaining: Include nuclear counterstain (e.g., DAPI) to visualize all cells

• Controls: Include positive controls (apoptosis-induced cells) and negative controls (primary antibody omitted)

For non-adherent cells, follow specialized protocols for suspension cells that may require additional steps for cell attachment to slides .

How can I quantify cleaved Caspase-3 levels in tissue samples using immunohistochemistry?

For quantification of cleaved Caspase-3 in tissue samples:

• Preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections at 4-6 μm thickness

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Antibody dilution: Use cleaved-CASP3 (D175) at 1:50-300 dilution

• Detection system: Use biotin-streptavidin-HRP or polymer-based detection systems

• Quantification methods:

  • Manual scoring: Evaluate percentage of positive cells and staining intensity

  • Digital image analysis: Use software to quantify positively stained cells

• Scoring systems:

  • Low/moderate/high expression categories based on percentage of positive cells

  • H-score calculation (combines intensity and percentage)

• Statistical analysis: Apply appropriate statistical tests based on experimental design

For research involving cancer tissues, categorize expression as high, moderate, low or absent according to established cut-offs for correlation with clinical outcomes .

Why might I observe discrepancies between the expected and actual molecular weight of cleaved Caspase-3 in Western blots?

Several factors can cause discrepancies in the observed molecular weight:

• Post-translational modifications: Phosphorylation or other modifications can alter protein migration

• Sample preparation: Incomplete denaturation or reduction can affect migration

• Different isoforms: Detection of different Caspase-3 isoforms

• Cleaved versus uncleaved form: The cleaved form (17-20 kDa) migrates differently than the full-length protein (32 kDa)

• Cross-reactivity: The antibody may detect related proteins in some species

• Degradation products: Additional fragments from protein degradation

The calculated MW of full-length Caspase-3 is approximately 32 kDa, but the observed band for the cleaved form is typically around 17-20 kDa, which represents the large subunit after proteolytic processing .

How can I distinguish between specific and non-specific staining when using the cleaved-CASP3 (D175) antibody?

To distinguish between specific and non-specific staining:

• Include proper controls:

  • Positive control: Tissues/cells known to express cleaved Caspase-3 (e.g., staurosporine-treated cells)

  • Negative control: Samples without primary antibody

  • Blocking peptide control: Pre-incubate antibody with blocking peptide

• Morphological assessment:

  • Specific staining should be consistent with expected subcellular localization (cytoplasmic)

  • Apoptotic cells show characteristic morphological changes (cell shrinkage, nuclear condensation)

• Pattern analysis:

  • Non-specific staining often appears as diffuse background or edge artifacts

  • Specific staining should be present in expected cell types and under appropriate conditions

• Validation experiments:

  • Confirm specificity using samples with CASP3 knockdown/knockout

  • Compare results with alternative antibodies targeting different epitopes

What potential cross-reactivity should I be aware of when using cleaved-CASP3 (D175) antibody across different species?

The cleaved-CASP3 (D175) antibody shows cross-reactivity with:

• Species reactivity: Validated for human, mouse, and rat samples

• Cross-reactivity with other caspases:

  • May detect cleaved DCP-1 (Drosophila caspase) due to sequence similarity

  • Potential detection of DRICE (Drosophila initiator caspase) after processing

  • Can detect Caspase-9-like activity in some experimental systems

• Considerations for non-mammalian systems:

  • In Drosophila, the antibody recognizes the tripeptide ETD epitope in different caspases

  • Validation experiments recommended when using in non-validated species

This cross-reactivity suggests the antibody may detect multiple components of the apoptotic pathway rather than exclusively Caspase-3 .

How can cleaved-CASP3 (D175) antibody be effectively used to study the temporal dynamics of apoptosis?

To study temporal dynamics of apoptosis using cleaved-CASP3 (D175) antibody:

• Time-course experiments:

  • Collect samples at multiple time points after apoptotic stimuli

  • Compare with other early/late apoptotic markers (e.g., phosphatidylserine externalization, PARP cleavage)

• Live-cell imaging approaches:

  • Combine with other fluorescent reporters of apoptosis

  • Use time-lapse microscopy to track individual cells

• Multiplexed detection systems:

  • Co-stain with markers for different phases of apoptosis

  • Use flow cytometry to quantify progression through apoptotic stages

• Experimental design table:

• Quantification methods:

  • Digital image analysis of immunofluorescence

  • Flow cytometry for population-level analysis

  • Western blot densitometry for relative quantification

What are the considerations for using cleaved-CASP3 (D175) antibody in different tissue contexts where background apoptosis rates vary?

For using cleaved-CASP3 (D175) antibody across different tissue contexts:

• Tissue-specific baseline determination:

  • Establish normal apoptotic rates for each tissue type

  • Use age-matched controls appropriate for developmental stage

• Optimization strategies for high-background tissues:

  • Titrate antibody concentration more carefully (1:50-1:300)

  • Modify blocking conditions (5-10% serum, 1-2 hours)

  • Consider alternative detection systems for signal amplification

• Specialized tissue considerations:

  • Highly proliferative tissues (intestine, skin): Higher baseline apoptosis

  • Neural tissues: Typically low baseline, may require signal enhancement

  • Embryonic tissues: Developmental apoptosis patterns vary by stage

  • Cancer tissues: Heterogeneous apoptotic responses

• Validation approaches:

  • Confirm with complementary apoptosis markers (TUNEL, Annexin V)

  • Use genetic models (caspase-3 knockout tissues as negative controls)

  • Employ tissue-specific positive controls (treated with known apoptotic stimuli)

How can cleaved-CASP3 (D175) antibody be used in combination with other markers to distinguish between apoptosis and other forms of cell death?

To distinguish between different cell death mechanisms:

• Multiplexed detection strategies:

  Death Pathway	Primary Marker	Secondary Markers	Cleaved-CASP3 Status
  Apoptosis (intrinsic)	Cleaved-CASP3	Cleaved-CASP9, Cytochrome c release	Positive
  Apoptosis (extrinsic)	Cleaved-CASP3	Cleaved-CASP8, Death receptor activation	Positive
  Necroptosis	MLKL phosphorylation	RIP1/RIP3 complex	Negative
  Pyroptosis	Cleaved-CASP1, IL-1β	NLRP3, ASC specks	Negative/Low
  Ferroptosis	Lipid peroxidation	GPX4 depletion, iron accumulation	Negative

• Immunofluorescence co-localization:

  • Co-stain with LC3B to differentiate from autophagy

  • Co-stain with RIP3 to identify necroptotic cells

  • Use TUNEL to confirm DNA fragmentation in apoptotic cells

• Morphological assessment:

  • Apoptosis: Cell shrinkage, nuclear condensation, membrane blebbing

  • Necrosis/Necroptosis: Cell swelling, membrane rupture

  • Pyroptosis: Cell swelling with membrane pores

• Functional validation:

  • Caspase inhibition tests (z-VAD-fmk should block cleaved-CASP3 signal)

  • Pathway-specific inhibitors to confirm mechanism

  • Genetic approaches (knockdown/knockout of key pathway components)

How does cleaved Caspase-3 expression compare to total Caspase-3 in tumor tissues, and what are the implications for cancer research?

Research comparing cleaved Caspase-3 to total Caspase-3 in cancer tissues reveals:

What are the best practices for using cleaved-CASP3 (D175) antibody in neurodegenerative disease research?

For neurodegenerative disease research:

• Tissue preparation considerations:

  • Post-mortem interval significantly affects cleaved Caspase-3 detection

  • Optimal fixation procedures for neural tissues (4% PFA, 24-48 hours)

  • Cryopreservation may better preserve epitopes compared to paraffin embedding

• Cell type-specific analysis:

  • Co-label with neural cell type markers (NeuN, GFAP, Iba1, etc.)

  • Distinguish between neuronal and glial apoptosis

  • Account for region-specific vulnerability to apoptosis

• Quantification approaches:

  • Stereological counting for unbiased quantification

  • Automated image analysis with machine learning algorithms

  • Relative quantification comparing affected vs. unaffected regions

• Model-specific considerations:

  Disease Model	Optimal Detection Method	Special Considerations
  Alzheimer's	IHC with DAB enhancement	Co-stain with Aβ/tau markers
  Parkinson's	Fluorescence multiplexing	Examine substantia nigra specifically
  ALS	Western blot + IF validation	Compare affected motor regions
  Stroke/Ischemia	Time-course analysis	Consider reperfusion timing

• Validation strategies:

  • Compare with other apoptotic markers (TUNEL, Annexin V)

  • Use genetic models (neuron-specific caspase manipulations)

  • Implement pharmacological interventions (caspase inhibitors)

How can quantitative analysis of cleaved Caspase-3 staining be standardized for reproducible research?

For standardized quantitative analysis:

• Standardized immunostaining protocol:

  • Fixed antibody concentration (optimal dilution determined by titration)

  • Consistent antigen retrieval methods

  • Automated staining platforms when possible

  • Include reference standard on each slide/batch

• Quantification systems:

  • Percentage of positive cells (0-100%)

  • Staining intensity scoring (0=negative, 1=weak, 2=moderate, 3=strong)

  • H-score calculation: Σ(intensity score × percentage)

  • Automated digital image analysis with validated algorithms

• Quality control measures:

  • Inter-observer variability assessment

  • Intra-assay and inter-assay coefficient of variation

  • Regular calibration with reference standards

  • Blinded assessment for research studies

• Reporting standards:

  • Detailed methodology reporting (antibody source, catalog number, dilution)

  • Description of scoring system and cut-off determinations

  • Raw data availability for meta-analysis

  • Statistical methods for comparing expression levels

• Meta-analysis considerations:

  • Hazard Ratios (HRs) with 95% confidence intervals for survival analyses

  • Assessment of heterogeneity using Q and I² tests

  • Fixed or random effect models based on heterogeneity levels

  • Evaluation of publication bias through funnel plots

How might single-cell approaches enhance our understanding of Caspase-3 activation dynamics beyond traditional antibody-based methods?

Emerging single-cell technologies offer new insights into Caspase-3 activation:

• Single-cell technologies:

  • Mass cytometry (CyTOF) for multiplexed protein detection

  • Single-cell RNA-seq to correlate transcript and protein levels

  • Live-cell biosensors for real-time caspase activity monitoring

  • Super-resolution microscopy for subcellular localization

• Advanced applications:

  • Spatial transcriptomics to map apoptosis in tissue context

  • In vivo imaging of caspase activity in animal models

  • Computational modeling of apoptotic signaling networks

  • Machine learning for pattern recognition in heterogeneous responses

• Comparative advantages over traditional methods:

  Method	Resolution	Multiplexing	Temporal Analysis	Limitations
  Cleaved-CASP3 IHC	Cellular	Limited (2-3 markers)	Endpoint only	Fixed samples
  Live-cell biosensors	Subcellular	Moderate (3-4 markers)	Continuous	Requires genetic modification
  CyTOF	Single-cell	High (30+ markers)	Endpoint only	Loses spatial information
  Spatial proteomics	Subcellular	High (40+ markers)	Endpoint only	Technically challenging

• Implementation challenges:

  • Need for specialized equipment and expertise

  • Higher costs compared to conventional antibody methods

  • Integration of multi-dimensional datasets

  • Standardization across different platforms