Cleaved-CASP3 (D175) Antibody

What is Cleaved-CASP3 (D175) and why is it important in apoptosis research?

Cleaved-CASP3 (D175) represents the activated form of caspase-3, which is created through proteolytic cleavage adjacent to aspartic acid residue 175. This cleaved form is the main executioner of apoptosis in cells. The significance of this protein lies in its central role in the apoptotic cascade, where it proteolytically cleaves and activates several proteins including caspase-6, caspase-7, and caspase-9. Additionally, at the onset of apoptosis, it cleaves poly(ADP-ribose) polymerase (PARP) at the '216-Asp-Gly-217' bond and is involved in the activation of sterol regulatory element binding proteins (SREBPs) .

From a methodological perspective, detecting cleaved caspase-3 provides a more accurate representation of apoptotic activity compared to measuring total caspase-3 levels, as it specifically identifies the active form involved in the execution phase of programmed cell death.

How does the D175 cleavage site differ from other caspase-3 cleavage points?

The D175 (Aspartic acid-175) site is the primary cleavage position that generates the active subunits of caspase-3. When caspase-3 is cleaved at D175, it generates two fragments: p17 (17 kDa) and p12 (12 kDa). This specific cleavage is crucial as it creates the active enzyme form .

The specificity of antibodies against this cleavage site is important because they can distinguish between the inactive zymogen (pro-caspase-3) and the active form. Methodologically, using antibodies specific to the D175 cleavage site allows researchers to directly monitor the activation process rather than just the presence of the protein.

How specific is the Cleaved-CASP3 (D175) antibody compared to other caspase antibodies?

The Cleaved-CASP3 (D175) antibody demonstrates high specificity for the active form of caspase-3. It recognizes endogenous levels of the large fragment (p17) of activated caspase-3 derived from cleavage adjacent to D175 and importantly, fails to recognize the full-length (inactive) caspase-3 or other cleaved caspases . This specificity makes it an excellent tool for distinguishing between inactive and active forms of caspase-3.

For experimental design, this means researchers can confidently use this antibody to quantify specifically the active form of caspase-3 without cross-reactivity concerns with the inactive zymogen or other caspase family members, allowing for more precise apoptosis detection.

What are the cross-reactivity profiles of Cleaved-CASP3 (D175) antibodies across species?

Cleaved-CASP3 (D175) antibodies typically demonstrate cross-reactivity across human, mouse, and rat samples . This cross-species reactivity is advantageous for comparative studies across different model systems. The conservation of the D175 cleavage site across these species allows for consistent detection using the same antibody.

When designing multi-species experiments, researchers should validate the antibody's performance in each species and application rather than assuming equivalent sensitivity across all models .

What are the optimal dilutions for different applications using Cleaved-CASP3 (D175) antibody?

For optimal results, researchers should perform a dilution series during initial experiments to determine the ideal concentration for their specific samples and detection methods . The high sensitivity of this antibody in ELISA applications (1:20000) makes it particularly suitable for quantitative measurements of cleaved caspase-3 in cell lysates.

How should samples be prepared to maximize detection of cleaved caspase-3?

Proper sample preparation is critical for accurate detection of cleaved caspase-3. For cell and tissue lysates:

• Harvest cells during peak apoptosis (typically 3-6 hours after apoptotic stimulus, depending on the model system)

• Use ice-cold lysis buffer containing protease inhibitors to prevent further enzymatic activity

• Include phosphatase inhibitors if phosphorylation status is relevant

• Maintain samples at 4°C during processing to minimize degradation

• Process samples quickly to capture the transient cleaved forms

For microscopy applications, fixation timing is crucial as cleaved caspase-3 signals can be lost if cells progress to late-stage apoptosis before fixation. Paraformaldehyde (4%) fixation for 15-20 minutes typically provides good antigen preservation for immunofluorescence and immunohistochemistry applications .

What controls should be included when using Cleaved-CASP3 (D175) antibody?

A robust experimental design requires appropriate controls:

• Positive control: Lysates from cells treated with known apoptosis inducers (e.g., staurosporine, TNF-α plus cycloheximide)

• Negative control: Lysates from healthy cells or cells treated with caspase inhibitors (e.g., Z-VAD-FMK)

• Antibody specificity control: Primary antibody omission or isotype control

• Cross-reactivity control: When validating across species, include samples from each species

• Loading control: For Western blots, include a housekeeping protein that remains stable during apoptosis (β-actin may not be ideal as it can be cleaved during late apoptosis)

Statistical analysis should include at least three biological replicates with appropriate significance testing (p < 0.05, p < 0.01, and p < 0.001) as standard practice for immunofluorescence quantification .

Why might the observed molecular weight of cleaved caspase-3 differ from the expected size?

These discrepancies can occur due to:

• Post-translational modifications affecting mobility

• Different modified forms appearing simultaneously in samples

• Gel concentration and running conditions altering apparent size

• Buffer composition affecting protein conformation

• The specific region recognized by the antibody

When interpreting Western blot results, focus on consistent band patterns rather than exact molecular weights, and confirm specificity with appropriate controls .

How can researchers distinguish between non-specific staining and true cleaved caspase-3 signal?

Distinguishing specific from non-specific signals requires methodical approaches:

• Morphological correlation: True cleaved caspase-3 positive cells should display apoptotic morphology (cell shrinkage, membrane blebbing, nuclear condensation)

• Co-staining validation: Use TUNEL or Annexin V staining in parallel sections or dual staining to confirm apoptotic status

• Nuclear/cytoplasmic localization: Early in apoptosis, cleaved caspase-3 appears primarily in the cytoplasm before translocation to various cellular compartments

• Dose-response relationship: Signal intensity should correlate with the strength of apoptotic stimuli

• Absorption controls: Pre-incubation of the antibody with the immunizing peptide should eliminate specific staining

For quantitative analysis in immunofluorescence, use appropriate thresholding methods and report data as mean ± standard deviation with statistical significance clearly indicated (p < 0.05, p < 0.01, p < 0.001) .

How can Cleaved-CASP3 (D175) antibody be used in multiplexed apoptosis assays?

Multiplexed apoptosis detection provides comprehensive analysis of the apoptotic cascade:

• Dual immunofluorescence: Combine cleaved caspase-3 antibodies with markers for:

  • Upstream initiators (cleaved caspase-8/9)

  • Downstream substrates (cleaved PARP)

  • Mitochondrial pathway components (cytochrome c)

  • Cell death outcomes (TUNEL)

• Flow cytometry applications:

  • Conjugate with compatible fluorophores that have minimal spectral overlap

  • Combine with Annexin V and propidium iodide to distinguish early vs. late apoptosis

  • Use in conjunction with cell cycle markers to correlate apoptosis with cell cycle phase

• Tissue microarray analysis:

  • Validate antibody performance across multiple tissue types

  • Establish quantitative scoring systems (percentage positive cells, staining intensity)

  • Correlate with clinical outcomes in pathological samples

When designing multiplexed assays, careful validation of antibody compatibility, spectral overlap, and staining protocols is essential to minimize false positive/negative results due to antibody cross-reactivity or fluorophore compensation issues.

What are the best approaches for quantifying cleaved caspase-3 levels in complex experimental systems?

Quantitative analysis of cleaved caspase-3 requires different approaches depending on the experimental context:

• In tissue sections and cellular imaging:

  • Use automated image analysis with consistent thresholding

  • Report both percentage of positive cells and staining intensity

  • Analyze multiple fields (typically ≥10) selected randomly

  • Use Z-stack imaging for thick specimens to avoid missing apoptotic cells

• In cell lysates:

  • ELISA provides more precise quantification than Western blotting

  • DuoSet IC ELISA kits offer high sensitivity for human and mouse samples

  • Normalize to total protein concentration

  • Use standard curves with known quantities of recombinant cleaved caspase-3

• For temporal dynamics:

  • Live-cell imaging with fluorescent reporter constructs

  • Time-course analysis with multiple sampling points

  • Correlation with other apoptotic events (phosphatidylserine externalization, nuclear condensation)

Statistical analysis should employ appropriate tests (t-test, ANOVA) with multiple comparisons correction when comparing across multiple groups or time points .

How does cleaved caspase-3 detection compare to other methods for assessing apoptosis?

Cleaved caspase-3 detection offers distinct advantages and limitations compared to other apoptosis assessment methods:

The ideal approach involves using cleaved caspase-3 detection in combination with at least one other method that detects a different aspect of the apoptotic process. This multiparameter approach provides stronger evidence of genuine apoptosis and helps distinguish it from other forms of cell death.

For quantitative studies, particularly when examining subtle differences in apoptotic responses, combining ELISA-based detection of cleaved caspase-3 with flow cytometric analysis of Annexin V/PI staining provides complementary data on both the enzymatic activation and the resulting cellular changes .