Cleaved-CASP5 (S331) Antibody

What is Cleaved-CASP5 (S331) Antibody and what cellular events does it detect?

Cleaved-CASP5 (S331) Antibody is a rabbit polyclonal antibody specifically designed to detect endogenous levels of the activated fragment of Caspase-5 p10 protein that results from proteolytic cleavage adjacent to the serine residue at position 331 (S331) . This antibody recognizes the cleaved form that emerges during caspase activation, not the inactive pro-enzyme form. The specificity of this antibody makes it particularly valuable for studying caspase activation cascades in inflammatory and apoptotic pathways. The antibody has been affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography, ensuring high specificity for the cleaved fragment .

What is the biological significance of Caspase-5 in cellular processes?

Caspase-5 (CASP5) is a member of the cysteine-aspartic acid protease family that plays central roles in programmed cell death (apoptosis) and inflammatory responses . As part of the execution phase of apoptosis, caspases exist as inactive proenzymes that undergo sequential proteolytic processing at conserved aspartic residues to produce two subunits (large and small) that dimerize to form the active enzyme . Beyond apoptosis, Caspase-5 has been implicated in non-canonical inflammasome activation where it cleaves CGAS (cyclic GMP-AMP synthase) and may play a significant role in regulating antiviral innate immune responses . The expression of CASP5 is regulated by interferon-gamma and lipopolysaccharide, highlighting its importance in immune signaling pathways .

How does cleavage at S331 affect Caspase-5 function?

The cleavage at serine 331 (S331) represents a critical proteolytic processing event that converts the inactive pro-caspase-5 into its active form. This site-specific cleavage results in the generation of the p10 fragment, which is essential for the formation of the functional caspase enzyme . The cleaved form exhibits proteolytic activity that the pro-enzyme lacks, enabling it to participate in inflammatory signaling cascades and potentially apoptotic pathways. The S331 cleavage site is located within the amino acid range 312-361, which is the region targeted by the immunogen used to generate the Cleaved-CASP5 (S331) antibody . Detection of this specific cleavage provides researchers with a precise marker for caspase-5 activation in biological samples.

What are the validated applications for Cleaved-CASP5 (S331) Antibody?

The Cleaved-CASP5 (S331) Antibody has been validated for Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . For Western Blot analysis, the recommended dilution ranges from 1:500 to 1:2000, allowing researchers to optimize based on their specific experimental conditions and detection system . For ELISA applications, a more dilute preparation of 1:20000 is recommended . The antibody has not yet been extensively tested in other applications such as immunohistochemistry, immunofluorescence, or flow cytometry, though these represent potential areas for method development by researchers working with this antibody. The antibody specifically detects human Caspase-5, making it suitable for research involving human cell lines and tissues .

What are the recommended protocols for Western Blot using this antibody?

For optimal Western Blot results with Cleaved-CASP5 (S331) Antibody, researchers should follow these methodological guidelines: First, prepare protein samples under reducing conditions and separate by SDS-PAGE. The cleaved Caspase-5 p10 fragment has a molecular weight of approximately 10 kDa , so appropriate percentage gels (15-20%) should be used for optimal resolution in this molecular weight range. After transfer to PVDF or nitrocellulose membrane, block with 5% non-fat milk or BSA in TBST. Incubate with primary antibody at dilutions between 1:500 and 1:2000 in blocking buffer overnight at 4°C . Following washing, use appropriate HRP-conjugated secondary antibodies and develop using enhanced chemiluminescence. For positive controls, consider using lysates from human cells treated with inflammatory stimuli known to activate inflammasomes, such as lipopolysaccharide combined with ATP or nigericin.

How should I optimize sample preparation for detection of cleaved Caspase-5?

Sample preparation is critical for successful detection of cleaved Caspase-5. Since the cleavage of Caspase-5 occurs during inflammasome activation or apoptotic signaling, researchers should consider incorporating appropriate stimulation protocols. Cell lysates should be prepared in the presence of protease inhibitors to prevent artifactual degradation of proteins. Additionally, phosphatase inhibitors may be valuable as the cleavage and activity of some caspases can be regulated by phosphorylation events. When working with tissue samples, rapid freezing and careful homogenization are essential to preserve protein integrity. Researchers should note that the antibody detects the endogenous levels of the fragment resulting from cleavage adjacent to S331 , so natural activation processes or specific inducers of inflammasome activation should be considered in experimental design. The cleaved p10 fragment runs at approximately 10 kDa on SDS-PAGE .

What are effective ways to induce Caspase-5 cleavage in experimental models?

To effectively induce Caspase-5 cleavage in experimental models, researchers should consider that Caspase-5 is primarily involved in inflammatory responses and is regulated by interferon-gamma and lipopolysaccharide (LPS) . A common approach involves priming cells with LPS (100-500 ng/ml for 3-6 hours) followed by exposure to inflammasome activators such as ATP (2-5 mM), nigericin (5-20 μM), or particulate matter like alum or silica. For non-canonical inflammasome activation, which may involve Caspase-5, intracellular LPS delivery via transfection or bacterial infection protocols can be effective. Additionally, since overexpression of the active form of Caspase-5 can induce apoptosis in fibroblasts , transfection with expression constructs encoding active Caspase-5 might serve as a positive control. Time course experiments are recommended to determine optimal time points for detecting cleaved Caspase-5, typically ranging from 30 minutes to 24 hours post-stimulation depending on the activator used.

Which cell types and tissues express significant levels of Caspase-5?

Caspase-5 expression is most prominent in immune cells and tissues involved in inflammatory responses. Monocytes, macrophages, dendritic cells, and other myeloid lineage cells typically express detectable levels of Caspase-5. Human peripheral blood mononuclear cells (PBMCs) are a reliable source of Caspase-5 expression, particularly when stimulated with interferon-gamma or LPS, which regulate CASP5 gene expression . Tissue-wise, Caspase-5 is expressed in lymphoid tissues including spleen and lymph nodes, as well as in intestinal epithelial cells that participate in mucosal immunity. Notably, Caspase-5 expression may be upregulated in inflammatory conditions and in certain cancer types. Researchers should be aware that Caspase-5 is a human protein, and the antibody specifically reacts with human samples , with no cross-reactivity validated for other species. This species specificity is an important consideration when designing experiments and selecting appropriate models.

How does inflammasome activation relate to Caspase-5 cleavage?

Inflammasome activation and Caspase-5 cleavage are intricately connected processes in the inflammatory response pathway. During non-canonical inflammasome activation, Caspase-5 undergoes proteolytic processing that results in the generation of the p10 subunit detected by the Cleaved-CASP5 (S331) Antibody . This cleavage event transforms the inactive pro-enzyme into its catalytically active form. Within this pathway, activated Caspase-5 cleaves CGAS (cyclic GMP-AMP synthase), potentially regulating antiviral innate immune responses . The cleavage adjacent to S331 is a specific marker of this activation process. While Caspase-1 is often considered the canonical inflammasome effector, Caspase-5 (along with Caspase-4) participates in non-canonical inflammasome pathways that respond to intracellular LPS and certain bacterial infections. Researchers investigating inflammasome biology should consider the temporal relationship between inflammasome assembly, Caspase-5 cleavage, and downstream events such as cytokine processing and pyroptosis.

What are common issues when detecting cleaved Caspase-5 and their solutions?

When working with Cleaved-CASP5 (S331) Antibody, researchers may encounter several technical challenges. One common issue is weak or absent signal, which may result from insufficient activation of Caspase-5 in the experimental system. This can be addressed by optimizing stimulation conditions or using positive controls where Caspase-5 cleavage is known to occur. Another challenge is the detection of multiple bands on Western blots. Since the cleaved p10 fragment has a relatively small size of 10 kDa , it may be difficult to distinguish from non-specific bands. Using gradient gels and optimizing transfer conditions for small proteins can improve resolution. Additionally, researchers may encounter issues with antibody specificity. The Cleaved-CASP5 (S331) Antibody is designed to detect the specific cleavage product adjacent to S331 , so other fragments or isoforms of Caspase-5 may not be recognized. For researchers experiencing high background, increasing blocking time or adjusting antibody dilution (1:500 to 1:2000 for WB) may improve signal-to-noise ratio.

How can I distinguish between specific and non-specific bands in Western Blot analysis?

Distinguishing specific cleaved Caspase-5 bands from non-specific signals requires careful experimental design and controls. The cleaved Caspase-5 p10 fragment should appear at approximately 10 kDa on SDS-PAGE . To confirm band specificity, researchers should implement several strategies: First, include a positive control such as lysates from cells treated with known inflammasome activators. Second, employ a negative control where Caspase-5 is not expected to be cleaved or activated. Third, consider using siRNA or CRISPR to knock down Caspase-5 expression; the specific band should disappear or be significantly reduced in such samples. Fourth, if available, use recombinant Caspase-5 protein as a size reference . Another approach is to perform a peptide competition assay, where pre-incubation of the antibody with the immunizing peptide should abolish specific binding. Finally, comparing band patterns between resting and stimulated cells can help identify the inducible bands that likely represent the cleaved form of Caspase-5.

What significance does the absence of cleaved Caspase-5 signal have in experimental settings?

The absence of a cleaved Caspase-5 signal in experimental settings warrants careful interpretation rather than immediate technical troubleshooting. This result may indicate that the experimental conditions have not triggered inflammasome activation or the specific proteolytic processing required for Caspase-5 cleavage at the S331 site . Biologically, this could suggest that the particular stimulus used does not engage pathways leading to Caspase-5 activation, or that regulatory mechanisms may be suppressing inflammasome assembly or caspase activation. Alternative interpretations include the possibility that Caspase-5 is being cleaved at sites other than S331, which would not be detected by this specific antibody. Researchers should consider temporal factors, as cleavage may occur transiently or at time points not captured in the experiment. To validate a true negative result, positive controls such as LPS plus ATP stimulation should be included, and the expression of total Caspase-5 should be confirmed using antibodies targeting the full-length protein .

How can Cleaved-CASP5 (S331) Antibody be integrated into inflammasome research?

The Cleaved-CASP5 (S331) Antibody offers valuable applications for advancing inflammasome research by providing a specific marker for non-canonical inflammasome activation . Researchers can employ this antibody to investigate the temporal dynamics of Caspase-5 activation in relation to other inflammasome components. By combining detection of cleaved Caspase-5 with assessments of other markers such as ASC speck formation, NLRP3 oligomerization, or Caspase-1 activation, researchers can develop comprehensive models of inflammasome assembly and activation sequences. The antibody also enables investigation of differential activation of canonical versus non-canonical inflammasome pathways in response to various pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). For mechanistic studies, researchers can use the antibody to assess how genetic or pharmacological manipulation of upstream signaling components affects Caspase-5 cleavage, potentially identifying novel regulators of inflammasome activation.

What is the relationship between Caspase-5 cleavage and CGAS regulation in antiviral responses?

Recent research has revealed a fascinating connection between Caspase-5 and cyclic GMP-AMP synthase (CGAS), linking inflammasome activation with antiviral innate immunity. During non-canonical inflammasome activation, Caspase-5 cuts CGAS and may play a significant role in regulating antiviral innate immune responses . The Cleaved-CASP5 (S331) Antibody provides researchers with a tool to investigate this relationship by monitoring Caspase-5 activation status. By conducting co-immunoprecipitation experiments with this antibody, researchers can explore the interaction between cleaved Caspase-5 and CGAS in various cellular contexts. Time-course experiments combining viral infection with detection of cleaved Caspase-5 can help establish the temporal relationship between inflammasome activation and antiviral signaling. Additionally, researchers can investigate how modulation of Caspase-5 activation affects downstream CGAS-STING pathway components, including IRF3 phosphorylation, type I interferon production, and interferon-stimulated gene expression.

What emerging roles is Caspase-5 playing in disease models and potential therapeutic applications?

Caspase-5's involvement in inflammasome activation and potential role in regulating antiviral responses positions it as an important molecule in various disease contexts. In inflammatory disorders, dysregulated Caspase-5 activation may contribute to pathological inflammation, making it a potential therapeutic target. The Cleaved-CASP5 (S331) Antibody enables researchers to monitor Caspase-5 activation status in patient samples and disease models . In infectious disease research, understanding how pathogens might inhibit or activate Caspase-5 could reveal immune evasion strategies. Additionally, given that overexpression of active Caspase-5 induces apoptosis in fibroblasts , there are potential implications for cancer research, particularly in understanding how cancer cells might evade inflammatory cell death. Researchers investigating Max, a component of the Myc/Max/Mad transcription regulation network important for cell growth, differentiation, and apoptosis, should note that Max is cleaved by Caspase-5, and this process requires Fas-mediated dephosphorylation of Max . This connection to transcriptional regulation opens avenues for exploring Caspase-5's role in cellular transformation and cancer biology.