Cleaved-CASP4 (Q81) Antibody

What is Cleaved-CASP4 (Q81) Antibody and what does it specifically detect?

Cleaved-CASP4 (Q81) Antibody is a polyclonal antibody that specifically detects endogenous levels of the activated Caspase-4 p20 protein fragment resulting from cleavage adjacent to Glutamine 81 (Q81). This antibody recognizes the cleaved form of human Caspase-4, a member of the cysteine-aspartic acid protease family that plays crucial roles in inflammatory responses, apoptosis, and cell migration . The antibody is generated against synthesized peptides derived from the internal region of human Caspase-4 p20, typically within the amino acid range 62-111 .

What are the primary applications for Cleaved-CASP4 (Q81) Antibody?

The Cleaved-CASP4 (Q81) Antibody has been validated for multiple research applications:

Optimal dilutions should be determined by researchers based on their specific experimental conditions .

What is the biological function of Caspase-4 in cellular processes?

Caspase-4 is an inflammatory caspase that:

• Acts as the effector of the non-canonical inflammasome by mediating lipopolysaccharide (LPS)-induced pyroptosis

• Indirectly activates the NLRP3 and NLRP6 inflammasomes

• Functions as a thiol protease that cleaves a tetrapeptide after an Asp residue, specifically catalyzing cleavage of CGAS, GSDMD, and IL18

• Is involved in the non-canonical inflammasome pathway independent of NLRP3 and CASP1

• Is activated by direct binding to LPS without requiring an upstream sensor

• Localizes primarily to the endoplasmic reticulum (ER), with association to the ER membrane requiring TMEM214

• Is involved in ER stress-induced apoptosis, a mechanism implicated in neurodegenerative conditions

How should I design Western blot experiments using Cleaved-CASP4 (Q81) Antibody?

For optimal Western blot results with Cleaved-CASP4 (Q81) Antibody:

• Sample preparation:

  • Prepare protein lysates from human cell lines or tissues using standard protocols

  • Include appropriate positive controls (such as 3T3 cells, which have been verified)

  • Consider including LPS-treated samples as positive controls for non-canonical inflammasome activation

• Technical parameters:

  • Load 20-40 μg of total protein per lane

  • Use a dilution ratio between 1:500 and 1:2000 of the antibody

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Select appropriate molecular weight markers to identify the cleaved fragment (expected bands at 43 kDa and 22 kDa)

• Data interpretation:

  • The observed molecular weight may show bands at 43 kDa (full-length) and 22 kDa (cleaved p20 subunit)

  • Note that the actual band pattern may not always be consistent with theoretical predictions due to post-translational modifications

How can I determine the optimal antibody concentration for my specific experimental conditions?

To determine optimal antibody concentration:

• Perform a titration experiment using a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000, 1:4000)

• Include appropriate positive and negative controls

• Assess signal-to-noise ratio for each dilution

• Select the dilution that provides the strongest specific signal with minimal background

• Consider that optimal dilutions may vary between applications (ELISA requires higher dilutions of 1:40000, while IHC typically uses 1:100-1:300)

• For each new lot of antibody or new experimental system, validation and optimization are recommended

What controls should I include when using Cleaved-CASP4 (Q81) Antibody?

For rigorous experimental design, include the following controls:

• Positive controls:

  • Cell lines verified to express Caspase-4 (such as 3T3 cells)

  • Cells treated with ER stress-inducing reagents, which are known to cleave Caspase-4

  • LPS-treated cells, which activate the non-canonical inflammasome pathway

• Negative controls:

  • Isotype control (rabbit IgG) to assess non-specific binding

  • Cell lines with CASP4 knockdown via siRNA

  • Tissues or cells from species other than human (as the antibody is human-specific)

• Technical controls:

  • Secondary antibody-only control to assess background

  • Blocking peptide competition assay to confirm specificity

  • Loading controls for Western blot (e.g., β-actin, GAPDH)

Why might I observe unexpected bands in Western blot analysis with Cleaved-CASP4 (Q81) Antibody?

Multiple factors can contribute to unexpected bands:

• Multiple protein forms:

  • Caspase-4 exists as inactive proenzymes that undergo proteolytic processing to generate large and small subunits

  • You may observe bands corresponding to both full-length (~43 kDa) and cleaved forms (~22 kDa)

• Post-translational modifications:

  • ADP-ribosylation by S. flexneri OspC3 can block CASP4 autoprocessing

  • Other modifications may alter migration patterns

• Technical issues:

  • Non-specific binding can occur if blocking or washing is insufficient

  • Degradation products may appear if samples are not properly handled

  • Cross-reactivity with related caspase family members might occur

• Sample preparation:

  • Inadequate lysis or incomplete protein denaturation

  • Protein overloading can lead to smearing or additional bands

To address these issues, optimize blocking conditions, sample preparation, and consider pre-adsorption of the antibody with the immunizing peptide to confirm specificity.

What are the critical storage and handling requirements for maintaining antibody activity?

To maintain optimal antibody performance:

• Storage conditions:

  • Store at -20°C for up to 12 months

  • Avoid repeated freeze/thaw cycles by preparing small aliquots upon receipt

  • The antibody is supplied in liquid form, typically in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Shipping and receipt:

  • The product is shipped with ice packs

  • Upon receipt, store immediately at the recommended temperature (-20°C)

  • Briefly centrifuge the vial to dislodge any liquid in the container's cap if necessary

• Working solution preparation:

  • Dilute immediately before use in appropriate buffer

  • Prepare only the amount needed for the current experiment

  • Keep on ice during handling

How does Caspase-4 activation correlate with different cellular stress conditions?

Caspase-4 activation patterns vary by stress condition:

• ER stress response:

  • Caspase-4 is specifically cleaved when cells are treated with ER stress-inducing reagents but not with other apoptotic reagents

  • Cleavage is not affected by Bcl-2 overexpression, suggesting a primary activation in ER stress-induced apoptosis rather than mitochondrial pathway activation

  • In neurodegenerative diseases like Alzheimer's, Caspase-4 is cleaved by amyloid-beta (Aβ) administration

• Bacterial infection and LPS response:

  • Caspase-4 is activated by direct binding to LPS during bacterial infection

  • Activation promotes GSDMD cleavage, followed by pyroptosis of infected cells

  • Mice lacking the encoded protein are resistant to endotoxic shock induced by LPS

• Inflammasome activation:

  • Caspase-4 acts as the effector of the non-canonical inflammasome independently of NLRP3 and CASP1

  • It promotes pyroptosis through GSDMD cleavage without involving secretion of cytokines IL1B and IL18

When interpreting Cleaved-CASP4 (Q81) Antibody results, consider these distinct activation patterns to properly contextualize your findings within specific stress response pathways.

What is the significance of detecting the cleaved form versus the full-length Caspase-4?

The distinction between cleaved and full-length Caspase-4 has important biological significance:

• Activation state:

  • Detection of the cleaved form (at Q81) indicates that Caspase-4 has been activated through proteolytic processing

  • Full-length Caspase-4 (~43 kDa) represents the inactive proenzyme form

• Functional implications:

  • Only the cleaved form is enzymatically active and capable of cleaving downstream substrates like GSDMD

  • Cleaved Caspase-4 is the functional effector in non-canonical inflammasome signaling

  • Ratio of cleaved to full-length protein can indicate the extent of pathway activation

• Pathological relevance:

  • Increased cleaved Caspase-4 has been associated with ER stress in neurodegenerative diseases

  • The cleaved form may serve as a biomarker for specific disease processes or therapeutic efficacy

Using antibodies specific to the cleaved form provides direct evidence of Caspase-4 activation rather than merely assessing expression levels, offering more precise insights into the inflammatory and stress response status of cells or tissues.

How can Cleaved-CASP4 (Q81) Antibody be used to study non-canonical inflammasome activation in infectious and inflammatory conditions?

For studying non-canonical inflammasome activation:

• Experimental design strategies:

  • Compare Caspase-4 cleavage patterns in cells exposed to various bacterial pathogens or purified LPS

  • Correlate Caspase-4 cleavage with downstream events such as GSDMD processing and pyroptosis

  • Use in combination with inhibitors of specific pathways to delineate signaling mechanisms

• Cell type considerations:

  • Focus on cells where the non-canonical inflammasome plays significant roles (macrophages, epithelial cells)

  • Compare primary cells versus cell lines to assess physiological relevance

  • Examine tissue-specific differences in non-canonical inflammasome activation

• Readout parameters:

  • Measure Caspase-4 cleavage by Western blot

  • Assess subcellular localization of cleaved Caspase-4 by immunofluorescence

  • Correlate with functional outcomes (cytokine release, cell death, inflammasome assembly)

  • Combine with gene knockdown approaches to establish causality

• Disease models:

  • Apply this approach to models of sepsis, inflammatory bowel disease, or other conditions where bacterial sensing is implicated

  • Compare findings between normal and disease states to identify potential therapeutic targets

What methodological approaches can be used to study Caspase-4's role in ER stress-induced apoptosis pathways?

To investigate Caspase-4's role in ER stress-induced apoptosis:

• Induction protocols:

  • Treat cells with established ER stress inducers (tunicamycin, thapsigargin, brefeldin A)

  • Compare effects with other apoptotic stimuli not associated with ER stress

  • Establish time courses to determine the kinetics of Caspase-4 cleavage relative to other ER stress markers

• Genetic manipulation approaches:

  • Perform siRNA-mediated knockdown of Caspase-4 to assess its necessity in ER stress-induced apoptosis

  • Overexpress wild-type or mutant forms of Caspase-4 to examine effects on sensitivity to ER stress

  • Use CRISPR/Cas9 to generate knockout models for more definitive functional studies

• Interaction studies:

  • Investigate the relationship between Caspase-4 and TMEM214, which is required for its association with the ER membrane

  • Examine crosstalk between Caspase-4 and other ER stress sensors (PERK, IRE1α, ATF6)

  • Assess potential interactions with Bcl-2 family proteins that regulate apoptosis

• Translational applications:

  • Apply these approaches to models of neurodegenerative diseases where ER stress is implicated

  • Examine Caspase-4 cleavage in response to amyloid-beta in Alzheimer's disease models

  • Develop therapeutic approaches targeting Caspase-4 activation in these conditions

How can researchers differentiate between the roles of Caspase-4 in apoptosis versus pyroptosis when interpreting experimental results?

Distinguishing Caspase-4's roles in different cell death pathways:

• Morphological assessment:

  • Apoptosis: cellular shrinkage, membrane blebbing, nuclear fragmentation

  • Pyroptosis: cellular swelling, membrane rupture, inflammatory release

• Biochemical markers:

  • Apoptosis-specific: measure cleavage of apoptotic caspases (Caspase-3, -7) and their substrates (PARP)

  • Pyroptosis-specific: assess GSDMD cleavage and pore formation

• Experimental approaches:

  • Conduct parallel experiments with specific inhibitors of apoptotic caspases versus Caspase-4

  • Use site-directed mutagenesis to generate Caspase-4 variants that selectively disrupt specific functions

  • Employ live-cell imaging with appropriate markers to distinguish death modalities in real-time

• Context-dependent analysis:

  • In ER stress conditions, focus on apoptotic readouts and correlation with other ER stress markers

  • In bacterial infection or LPS stimulation, emphasize pyroptotic outcomes and inflammatory markers

  • Consider that these pathways may not be mutually exclusive in all cellular contexts

By systematically applying these approaches, researchers can more accurately attribute observed phenotypes to specific Caspase-4 functions in different cellular death and inflammatory pathways.

How can Cleaved-CASP4 (Q81) Antibody data be integrated with other -omics approaches to understand inflammatory pathways comprehensively?

For multi-omics integration strategies:

• Complementary techniques:

  • Proteomics: Use mass spectrometry to identify Caspase-4 cleavage products and interaction partners

  • Transcriptomics: Correlate Caspase-4 activation with global gene expression changes

  • Metabolomics: Assess metabolic changes associated with different modes of cell death triggered by Caspase-4

• Data integration workflow:

  • Use Cleaved-CASP4 (Q81) Antibody to establish activation status in experimental conditions

  • Perform parallel -omics analyses on the same samples

  • Apply computational approaches to identify correlations and causal relationships

  • Validate key findings with targeted functional assays

• Systems biology approaches:

  • Map Caspase-4 activation onto known inflammatory and cell death pathway networks

  • Identify novel connections and feedback mechanisms

  • Model the temporal dynamics of pathway activation

This integrated approach provides a more comprehensive understanding of Caspase-4's role within the broader cellular response systems and may identify novel therapeutic targets or biomarkers.

What are the most reliable quantification methods for analyzing Cleaved-CASP4 (Q81) signal in different experimental platforms?

For accurate quantification:

• Western blot quantification:

  • Use appropriate loading controls (β-actin, GAPDH) for normalization

  • Apply densitometry analysis using standardized software (ImageJ, Image Lab)

  • Calculate the ratio of cleaved to full-length Caspase-4 for more meaningful comparison

  • Include a standard curve using recombinant protein for absolute quantification

• ELISA-based quantification:

  • Follow the recommended 1:40000 dilution for optimal sensitivity and specificity

  • Generate standard curves with purified recombinant proteins

  • Include spike-in controls to assess recovery in complex samples

  • Apply appropriate statistical analyses to determine significance

• Immunofluorescence quantification:

  • Use standardized image acquisition parameters

  • Apply automated analysis algorithms to prevent bias

  • Quantify nuclear versus cytoplasmic localization

  • Present data as mean fluorescence intensity or percentage of positive cells