Cleaved-CASP6 (D162) Antibody

What is Cleaved-CASP6 (D162) Antibody?

Cleaved-CASP6 (D162) is a polyclonal antibody that specifically detects endogenous levels of fragments of activated Caspase-6 p18 protein resulting from cleavage adjacent to Aspartic acid at position 162 (D162). This antibody recognizes the neoepitope created when Caspase-6 is activated through proteolytic processing, making it a valuable tool for detecting active Caspase-6 in experimental systems .

What are the common applications for Cleaved-CASP6 (D162) Antibody?

The antibody is utilized in multiple research applications including Western Blot (WB) at dilutions of 1:500-1:2000, Immunohistochemistry (IHC) at 1:100-1:300, Immunofluorescence (IF) at 1:50-200, and ELISA at 1:10000 . These applications allow researchers to detect activated Caspase-6 in various experimental contexts, from protein lysates to fixed tissue sections, providing versatility in apoptosis research and neurodegenerative disease studies .

What species reactivity does the antibody demonstrate?

The Cleaved-CASP6 (D162) antibody primarily demonstrates reactivity with mouse and rat samples . When designing experiments, researchers should note these species limitations and validate reactivity when working with samples from other species. Cross-reactivity with human samples may be possible but should be empirically verified before conducting critical experiments .

How is the antibody stored and what is its formulation?

The antibody is provided as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . For optimal stability and activity, it should be stored at -20°C for up to one year from receipt . To preserve antibody integrity, aliquoting is recommended to avoid repeated freeze-thaw cycles that could degrade the antibody and reduce its effectiveness in experimental applications .

How does Cleaved-CASP6 (D162) antibody differentiate between inactive procaspase-6 and activated caspase-6?

The Cleaved-CASP6 (D162) antibody specifically recognizes the neoepitope exposed after proteolytic processing at Asp162, which occurs during caspase activation. This allows researchers to distinguish between the inactive zymogen (procaspase-6) and the activated form in experimental systems . The antibody detects endogenous levels of activated Caspase-6 p18 protein fragments resulting from this specific cleavage event, providing a precise tool for monitoring caspase activation in apoptotic pathways .

What is the role of Caspase-6 in tau pathology and neurodegenerative diseases?

Caspase-6 has emerged as a significant player in neurodegenerative disorders through its ability to cleave tau protein. Tau truncation (tr-tau) by active Caspase-6 (aCasp-6) generates tau fragments that may be toxic in diseases like Alzheimer's . Research using this antibody has revealed that aCasp-6 levels show early and progressive increases in Alzheimer's disease, suggesting that Caspase-6 activation extends beyond executing apoptosis and may contribute to pathological tau cleavage . Therapeutic modulation of Caspase-6 activity represents a potential strategy for intervention in tauopathies .

What is the relationship between Caspase-6 and alternative splicing regulation?

Caspase-6 has been identified as a novel regulator of alternative RNA splicing, representing a function distinct from its canonical role in apoptosis . Proteomic studies have revealed significant changes in Caspase-6 levels (showing a normalized H/L ratio of 0.30312) in experiments examining splicing regulation . This discovery expands our understanding of Caspase-6 functions beyond cell death pathways, suggesting broader roles in cellular regulatory processes that may be relevant to both normal physiology and disease states .

How can I validate the specificity of Cleaved-CASP6 (D162) antibody in my experiments?

To validate antibody specificity, researchers should implement peptide competition assays using the synthesized immunogenic peptide. Multiple search results demonstrate this approach, showing that antibody binding can be blocked by pre-incubation with the synthesized peptide . Additionally, positive controls such as lysates from HeLa cells treated with apoptosis inducers like Etoposide (25μM for 60 minutes) are effective for validating antibody performance in detecting cleaved Caspase-6 . Western blot analysis should reveal bands at approximately 28+35kDa corresponding to Caspase-6 cleavage products .

What are optimal sample preparation methods for detecting cleaved Caspase-6?

For optimal detection of cleaved Caspase-6, cell lysates should be prepared from apoptotic cells (ideally treated with known apoptosis inducers like Etoposide) . For tissue samples, proper fixation is critical – researchers typically use formalin-fixed, paraffin-embedded sections for immunohistochemistry applications . When performing multiplex immunofluorescence studies, a sequential staining protocol may be necessary, as demonstrated in studies examining the relationship between active Caspase-6, truncated tau, and hyperphosphorylated tau in neurodegenerative diseases .

How can I implement multiplex immunofluorescence to study Caspase-6 alongside other apoptotic or neurodegenerative markers?

Based on advanced research protocols, a five-plex immunofluorescence approach can be implemented to simultaneously detect active Caspase-6, tau truncation, and phosphorylated tau in the same tissue sections . This methodology requires careful antibody selection to avoid cross-reactivity, sequential staining with appropriate elution steps between antibody applications, and signal development using distinct fluorophores for each target . Additionally, autofluorescence reduction steps (such as Sudan Black B treatment) are essential to enhance signal specificity, particularly when working with brain tissue samples .

What are the common issues in Western blot applications of Cleaved-CASP6 (D162) antibody and how can they be resolved?

When performing Western blots with the Cleaved-CASP6 (D162) antibody, researchers may encounter weak or absent signals even in positive control samples. To resolve this issue, ensure proper induction of apoptosis in positive control samples (such as treatment with Etoposide 25μM for 60 minutes) . Optimization of antibody concentration is also critical; while the recommended dilution range is 1:500-1:2000 for Western blot, researchers may need to adjust this based on their specific experimental conditions . Additionally, using fresh samples and adding protease inhibitors during lysate preparation helps preserve the cleaved forms of Caspase-6 that might otherwise degrade rapidly .

How can I improve signal-to-noise ratio in immunohistochemistry applications?

To improve signal-to-noise ratio in IHC applications, careful optimization of antibody dilution (recommended range: 1:100-1:300) is essential . Background reduction can be achieved through thorough blocking steps using appropriate blocking reagents (typically BSA or serum from the species of the secondary antibody) . For specific detection of cleaved Caspase-6 in tissue samples, implementing an antigen retrieval step (heat-induced epitope retrieval in citrate buffer pH 6.0) can significantly enhance epitope accessibility and improve specific binding . When using fluorescence detection methods, treatment with Sudan Black B (0.8% in 70% ethanol) effectively reduces tissue autofluorescence, particularly in brain samples .

What controls should be included when using Cleaved-CASP6 (D162) antibody in neurodegenerative disease research?

When studying neurodegenerative diseases, researchers should include several critical controls: (1) age-matched healthy control tissues to establish baseline Caspase-6 activation levels, (2) positive control tissues from conditions known to exhibit Caspase-6 activation, (3) peptide competition controls to confirm antibody specificity, and (4) appropriate isotype controls to assess non-specific binding . In studies examining multiple tauopathies, including samples from different diseases (such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and argyrophilic grain disease) provides important comparative insights into disease-specific patterns of Caspase-6 activation .

How does the detection of cleaved Caspase-6 inform our understanding of neurodegeneration progression?

Detection of cleaved Caspase-6 using the D162 antibody has revealed that active Caspase-6 shows early and progressive increases in Alzheimer's disease, suggesting roles beyond the execution phase of apoptosis . This finding supports the hypothesis that Caspase-6 activation contributes to pathological tau cleavage throughout disease progression . Quantification of cleaved Caspase-6 in neurons and astroglia across different tauopathies can provide insights into disease-specific mechanisms and potential therapeutic targets. The co-occurrence of active Caspase-6 with truncated tau and hyperphosphorylated tau in the same cells suggests a mechanistic relationship between these pathological events that could be leveraged for therapeutic intervention .

What are the implications of Caspase-6's role in alternative splicing for disease mechanisms?

The identification of Caspase-6 as a regulator of alternative splicing opens new avenues for understanding disease mechanisms beyond apoptosis . This function suggests that Caspase-6 may influence gene expression patterns that contribute to disease phenotypes even before triggering cell death. In neurodegenerative diseases where aberrant RNA processing has been implicated, Caspase-6's role in splicing regulation could represent an early disease mechanism that precedes overt neurodegeneration . This functional duality positions Caspase-6 as both a mediator of disease progression through tau cleavage and a potential regulator of disease-associated gene expression changes through splicing modulation .