Cleaved-CASP3 (A175) Antibody

What is Cleaved-CASP3 (Asp175) Antibody and what specific epitope does it recognize?

Cleaved-CASP3 (Asp175) Antibody specifically recognizes the cleaved form of Caspase-3 at the aspartic acid residue at position 175. During apoptosis, Caspase-3 undergoes proteolytic cleavage between Asp175 and Ser176, separating the large and small subunits. This cleavage exposes the epitope that is detected by the antibody . The antibody does not recognize unprocessed Caspase-3, making it a specific marker for the activated form of this key executioner caspase in the apoptotic pathway .

Research has demonstrated that the epitope recognized in apoptotic cells is specifically ETD (glutamic acid-threonine-aspartic acid), as shown by blocking experiments with synthetic peptides . This ability to exclusively detect the cleaved, active form makes this antibody valuable for distinguishing between latent and activated apoptotic processes in experimental systems.

What applications is Cleaved-CASP3 (Asp175) Antibody validated for?

The Cleaved-CASP3 (Asp175) Antibody has been validated for multiple experimental applications:

The antibody has been extensively used in various experimental systems, including cell lines treated with apoptosis inducers like staurosporine, tissue sections from different organs, and clinical samples from cancer studies .

What is the species cross-reactivity of Cleaved-CASP3 (Asp175) Antibody?

The Cleaved-CASP3 (Asp175) Antibody shows confirmed reactivity with:

• Human

• Mouse

• Rat

• Monkey (Mk)

Some commercially available antibodies are specifically designed for human samples only , while others have broader cross-reactivity . When using this antibody with species not explicitly listed by the manufacturer, validation is strongly recommended through appropriate positive controls .

The cross-reactivity is based on sequence homology at the Asp175 cleavage site region of Caspase-3, which is highly conserved across mammalian species. This conservation allows the same antibody to be used in comparative studies across different model organisms .

What are the optimal sample preparation methods for detecting cleaved caspase-3?

For Western Blotting:

• Harvest cells at appropriate timepoints after apoptosis induction (e.g., 4 hours after staurosporine treatment for Jurkat cells)

• Lyse cells in a buffer containing protease inhibitors to prevent further processing

• Determine protein concentration and load equal amounts (typically 20-50 μg)

• Use reducing conditions with Immunoblot Buffer Group 3 for optimal results

• Transfer to PVDF membrane for best detection of the 17-19 kDa cleaved fragments

For Immunohistochemistry/Immunofluorescence:

• Fix tissues or cells quickly to preserve the cleaved epitope (4% paraformaldehyde is common)

• For paraffin sections, heat-mediated antigen retrieval with pH 9.0 EDTA buffer is recommended

• Block with appropriate blocking solution to reduce background

• Incubate with primary antibody (1:400 dilution) overnight at 4°C or for 1-3 hours at room temperature

• For fluorescence detection, anti-rabbit secondary antibodies conjugated to fluorophores such as NorthernLights™ 557 have been successfully used

The timing of sample collection is critical, as cleaved caspase-3 appears transiently during apoptosis, typically peaking 3-4 hours after apoptotic stimulus in cell culture systems .

What controls should be included when using Cleaved-CASP3 (Asp175) Antibody?

Positive Controls:

• Jurkat human acute T cell leukemia cell line treated with 1 μM staurosporine for 4 hours

• DA3 mouse myeloma cell line treated with staurosporine

• Tissue sections known to contain apoptotic cells (e.g., developing thymus)

• Gamma herpesvirus 68-infected alveolar epithelial cells (for mouse studies)

Negative Controls:

• Untreated Jurkat or DA3 cells

• Primary antibody omission

• Blocking with the specific peptide containing the ETD epitope

• Tissues or cells from caspase-3 knockout models (if available)

Validation Controls:

• Parallel staining for cleaved PARP (Asp214), another marker of apoptosis that is a substrate of active caspase-3

• Western blot analysis comparing cleaved caspase-3 detection with total caspase-3 antibodies

• Use of caspase inhibitors (e.g., Z-VAD-FMK) to confirm specificity to apoptotic mechanisms

Including these controls helps validate the specificity of the signal and ensures that the observed staining truly represents activated caspase-3 during apoptosis rather than non-specific binding or artifacts.

What are the expected band sizes in Western blotting and how should results be interpreted?

When using Cleaved-CASP3 (Asp175) Antibody in Western blotting, researchers should expect the following:

Expected Band Sizes:

• 17-19 kDa bands representing the cleaved large subunit of caspase-3

• Some antibodies may detect both the p17 and p12 subunits (17-19 kDa and 12 kDa respectively)

• The uncleaved procaspase-3 (32 kDa) should NOT be detected by cleaved-specific antibodies

Interpretation Guidelines:

• Presence of 17-19 kDa bands indicates activation of caspase-3 through proteolytic cleavage

• Band intensity correlates with the degree of apoptosis in the sample

• Time-course experiments often show increasing intensity of cleaved bands with prolonged apoptotic stimulus

• Multiple bands within the 17-19 kDa range may represent different cleavage products or post-translational modifications

• No bands in positive control samples may indicate technical issues with antibody or protocol

It's important to note that in some cases, the observed molecular weight may differ slightly from expectations due to factors such as post-translational modifications or differences in electrophoresis conditions . When comparing samples, quantification should be normalized to appropriate loading controls.

How can Cleaved-CASP3 (Asp175) Antibody be used to distinguish between apoptosis and other forms of cell death?

Cleaved-CASP3 (Asp175) Antibody is highly specific for apoptotic cell death but has limitations in distinguishing between different cell death modalities:

Methodological Approach:

• Multiple marker strategy: Combine cleaved caspase-3 detection with other apoptosis markers (TUNEL, Annexin V, cleaved PARP) and markers of alternative death pathways (RIPK1/3 for necroptosis, LC3 for autophagy)

• Inhibitor studies: Use specific inhibitors (Z-VAD-FMK for apoptosis, Necrostatin-1 for necroptosis) alongside cleaved caspase-3 detection

• Morphological correlation: Correlate cleaved caspase-3 immunostaining with morphological features of apoptosis (chromatin condensation, membrane blebbing) using electron or high-resolution microscopy

Important Considerations:

• The anti-cleaved caspase-3 antibody specifically recognizes the Asp175 residue but may not discriminate between active caspase-3 and cleaved caspase-3 generated by other proteases such as calpains

• To confirm that active caspase-3 is being measured, parallel staining for cleaved PARP (a direct substrate of active caspase-3) is recommended

• In some pathological contexts, mixed forms of cell death can occur simultaneously, complicating interpretation

Research has shown that cleaved caspase-3 staining is most reliable when used within a panel of complementary apoptosis markers rather than as a standalone indicator of apoptotic cell death.

What is the relationship between stromal versus tumor cell cleaved caspase-3 expression and clinical outcomes?

Research has revealed intriguing differences in the prognostic significance of cleaved caspase-3 expression depending on its cellular localization in cancer tissues:

Key Research Findings:

• High levels of cleaved caspase-3 within colorectal tumor-associated stroma correlate with good prognosis, functioning as an independent marker of favorable outcomes

• This contrasts with cleaved caspase-3 expression within tumor cells, which has more variable prognostic implications

• The tumor microenvironment's apoptotic activity appears to modulate tumor progression through mechanisms involving immune surveillance and stromal remodeling

Methodological Approach for Assessment:

• Tissue microarray (TMA) analysis with cleaved caspase-3 immunohistochemistry

• Quantitative scoring of both intensity and distribution of staining

• Separate evaluation of stromal versus epithelial (tumor) compartments

• Correlation with clinical outcome data using multivariate analysis to control for other prognostic factors

This research highlights the importance of distinguishing between stromal and tumoral cleaved caspase-3 expression when evaluating its significance in cancer tissues. The pro-apoptotic activity in the tumor microenvironment may contribute to anti-tumor immunity and improved clinical outcomes.

How can blocking peptide experiments be designed to validate cleaved caspase-3 antibody specificity?

Blocking peptide experiments are crucial for confirming antibody specificity, particularly in immunohistochemistry and immunofluorescence applications:

Experimental Design:

• Peptide selection: Use synthetic peptides corresponding to the epitope recognized by the cleaved caspase-3 antibody (ETD sequence) and control peptides with different sequences

• Pre-incubation protocol:

  • Mix the cleaved caspase-3 antibody with blocking peptide (typically 5-10 fold molar excess)

  • Incubate for 60 minutes at room temperature or 4°C before applying to samples

  • In parallel, prepare antibody without peptide or with control peptides

• Validation criteria:

  • Complete abolishment of immunoreactivity with specific blocking peptide

  • Maintained immunoreactivity with control peptides

  • Consistent results across multiple sample types

Research Application Example:
In Drosophila eye imaginal discs, blocking experiments with peptide A (containing the ETD epitope) completely abolished cleaved caspase-3 antibody immunoreactivity in both GMR-hid and dcp-1 drICE double mutant GMR-hid eye discs. In contrast, peptide B and control peptides C and D failed to block immunoreactivity, demonstrating that the antibody specifically detected the ETD epitope in apoptotic cells .

This methodological approach provides robust validation of antibody specificity and helps researchers distinguish between true apoptosis signaling and potential cross-reactivity with other epitopes.

What considerations should be made when using Cleaved-CASP3 (Asp175) Antibody in ELISA versus immunoblotting formats?

The detection of cleaved caspase-3 can be performed using different methodological platforms, each with unique considerations:

ELISA-Based Detection:

• Assay principle: Sandwich immunoassay with capture antibody specific for cleaved caspase-3 (Asp175) and detection antibody against total caspase-3

• Advantages:

  • Quantitative measurement with higher throughput

  • Greater sensitivity for detecting low levels of cleaved caspase-3

  • Reduced processing time compared to Western blotting

• Sample considerations:

  • Requires whole cell lysates prepared with specific lysis buffers

  • Protein concentration standardization is critical

  • Sample dilution series recommended for optimal detection range

Western Blotting Detection:

• Assay principle: Direct visualization of cleaved caspase-3 fragments by molecular weight

• Advantages:

  • Visual confirmation of specific band size (17-19 kDa)

  • Ability to detect multiple cleavage products simultaneously

  • Useful for confirming antibody specificity

• Technical considerations:

  • Reducing conditions required for optimal detection

  • PVDF membrane recommended over nitrocellulose

  • Enhanced chemiluminescence detection provides best sensitivity

Comparative Data:
Data from MSD MULTI-ARRAY Cleaved Caspase-3 Assay shows increased signal with titration of cleaved caspase-3 positive cell lysate (staurosporine-treated Jurkat cells), while signal for negative lysate remains low throughout the titration. This pattern correlates well with Western blot analysis using the same samples, demonstrating that ELISA-based methods provide quantitative measures comparable to traditional Western blotting but with higher throughput potential .

What are common causes of false-positive and false-negative results when using Cleaved-CASP3 (Asp175) Antibody?

False-Positive Results - Causes and Solutions:

False-Negative Results - Causes and Solutions:

Methodological Validation:
To determine whether negative staining truly represents absence of apoptosis, researchers should include positive controls (staurosporine-treated Jurkat cells) in every experiment and consider using complementary methods like TUNEL assay or Annexin V staining to confirm results from multiple methodological approaches .

How can signal-to-noise ratio be optimized when using Cleaved-CASP3 (Asp175) Antibody in different applications?

Optimizing signal-to-noise ratio is critical for accurate detection of cleaved caspase-3 across different experimental platforms:

For Western Blotting:

• Blocking optimization: 5% non-fat dry milk in TBST generally provides optimal blocking

• Antibody dilution: Start with 1:1000 and adjust based on signal intensity

• Washing stringency: Multiple TBST washes (4-5 times, 5 minutes each) minimize background

• Enhanced chemiluminescence reagents: Use high-sensitivity ECL substrates for weak signals

• Exposure time optimization: Capture multiple exposures to find optimal signal-to-noise ratio

For Immunohistochemistry/Immunofluorescence:

• Antigen retrieval: Heat-mediated retrieval with pH 9.0 EDTA buffer enhances specific signal

• Blocking serum selection: Use serum from the species in which secondary antibody was raised

• Antibody concentration: 1:400 dilution is recommended starting point

• Incubation conditions: Overnight at 4°C generally provides better signal-to-noise than shorter incubations

• Secondary antibody selection: High-affinity, minimal cross-reactivity antibodies reduce background

• Counterstaining optimization: DAPI concentration affects background in fluorescence applications

For ELISA-Based Detection:

• Blocking solution optimization: Blocking solution-A provided in commercial kits minimizes background

• Sample dilution: Create dilution series to find optimal working range

• Detection antibody concentration: Follow kit guidelines precisely

• Washing thoroughness: Three complete washes with Tris Wash Buffer between steps

• Read buffer composition: 1X Read Buffer T provides optimal electrochemiluminescent signal

Systematic optimization of these parameters for each specific application and sample type is essential for achieving reliable and reproducible results with cleaved caspase-3 antibodies.

What is the significance of different cleavage sites in caspase-3 and their detection by specific antibodies?

Caspase-3 undergoes complex proteolytic processing that generates multiple cleavage products with distinct functional significance:

Major Cleavage Sites and Their Significance:

• Asp175 (primary focus of most antibodies):

  • Separates the large (p17) and small (p12) subunits

  • Critical for activation of enzymatic activity

  • Generated primarily by initiator caspases (caspase-8, caspase-9)

  • Most reliable marker of canonical apoptosis pathway activation

• Other cleavage sites:

  • Asp9/Asp28: N-terminal processing that may affect subcellular localization

  • Asp176: Alternative processing site that affects antibody recognition

  • Additional sites generated by non-caspase proteases during non-apoptotic events

Methodological Implications:

• Antibodies specific to different cleavage sites may yield different staining patterns

• For comprehensive analysis of caspase-3 activation, combining antibodies that recognize different epitopes can provide complementary information

• When comparing results across studies, attention to the specific cleavage site recognized by the antibody is essential

• Non-caspase proteases (e.g., calpains, cathepsins) can generate caspase-3 fragments that may or may not be detected by Asp175-specific antibodies

Understanding these distinctions is crucial for accurate interpretation of experimental results, particularly in complex biological contexts where multiple proteolytic pathways may be simultaneously activated.

How is Cleaved-CASP3 (Asp175) Antibody being used in cancer research and prognostication?

Cleaved caspase-3 detection has become an important tool in cancer research for understanding tumor biology and predicting patient outcomes:

Current Research Applications:

• Prognostic biomarker development:

  • High stromal cleaved caspase-3 levels correlate with good prognosis in colorectal cancer

  • The relationship between tumor cell apoptosis and patient outcomes varies by cancer type

  • Quantitative scoring systems being developed to standardize assessment

• Therapeutic response monitoring:

  • Measurement of cleaved caspase-3 levels to assess efficacy of pro-apoptotic cancer therapies

  • Dynamic changes in cleaved caspase-3 expression during treatment serve as pharmacodynamic markers

  • Combination with proliferation markers provides comprehensive tumor response assessment

• Tumor microenvironment characterization:

  • Differential patterns of apoptosis in tumor versus stromal compartments

  • Co-localization with immune cell markers to understand immune-mediated tumor cell killing

  • Relationship between apoptotic indices and angiogenesis or hypoxia markers

Methodological Approaches:

• Tissue microarray analysis with automated quantitative scoring

• Multiplex immunofluorescence to correlate cleaved caspase-3 with other biomarkers

• Combination of cleaved caspase-3 with cleaved PARP detection for confirmation

• Integration of apoptotic indices with clinical outcome data using multivariate analysis

This research highlights the complex role of apoptosis in cancer progression and the importance of cellular context when interpreting cleaved caspase-3 expression patterns in tumor tissues.

What are the considerations for using Cleaved-CASP3 (Asp175) Antibody in neurodegenerative disease research?

Cleaved caspase-3 detection has important applications in neurodegenerative disease research, with several unique considerations:

Research Applications in Neurodegenerative Contexts:

• Non-apoptotic functions in neurons:

  • Cleaved caspase-3 has been implicated in synaptic plasticity and neurite pruning

  • Low-level activation may occur without triggering complete apoptosis

  • Detection requires high-sensitivity methods due to low abundance

• Disease-specific considerations:

  • Alzheimer's disease: Caspase-3 cleavage of tau protein generates pathological fragments

  • Huntington's disease: Caspase-3 involved in the cleavage of huntingtin protein

  • Parkinson's disease: Selective vulnerability of dopaminergic neurons to caspase-3 activation

Methodological Approaches:

• Tissue preparation optimization:

  • Rapid fixation essential to preserve transient cleaved caspase-3 in neural tissues

  • Antigen retrieval conditions more critical than in other tissues

  • Background reduction techniques particularly important due to high lipid content

• Detection strategies:

  • Dual labeling with neuronal markers and cleaved caspase-3

  • Co-localization analysis with disease-specific protein aggregates

  • Quantitative analysis of subcellular localization (nuclear vs. cytoplasmic vs. neurites)

  • Serial sectioning to follow cleaved caspase-3 expression along neuronal processes

• Validation approaches:

  • Confirmation with multiple antibodies recognizing different epitopes

  • Functional assays measuring caspase-3 enzymatic activity

  • Correlation with other markers of neuronal stress or death