CASP10 Antibody

What is CASP10 and what is its role in cellular processes?

CASP10 (Caspase-10) plays a critical role in the activation cascade of caspases responsible for apoptosis execution. It is recruited to both Fas- and TNFR-1 receptors in a FADD-dependent manner and may participate in the granzyme B apoptotic pathways. Functionally, CASP10 cleaves and activates several effector caspases including CASP3, CASP4, CASP6, CASP7, CASP8, and CASP9 . This proteolytic activity is essential for programmed cell death, with CASP10 acting as an initiator caspase in death receptor signaling pathways. Understanding its function provides context for antibody-based detection methods in apoptosis research.

What types of CASP10 antibodies are available for research?

Researchers have access to several types of CASP10 antibodies, with rabbit polyclonal antibodies being among the most commonly utilized. These antibodies are designed to recognize specific epitopes within the CASP10 protein. For instance, some antibodies target synthetic peptides corresponding to regions within Human CASP10 amino acid 500 to the C-terminus . When selecting an antibody, researchers should consider the specific isoform detection needs, as CASP10 exists in multiple isoforms (including isoform 7 and isoform C) with distinct functional properties .

What are the common applications for CASP10 antibodies in research?

CASP10 antibodies are validated for multiple experimental applications including:

• Western blot (WB): Used to detect CASP10 protein expression levels and processing

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies

• Immunohistochemistry (IHC): To examine tissue expression patterns

• Immunoprecipitation (IP): For protein-protein interaction studies

Each application requires specific optimization parameters. For instance, Western blot applications typically use a dilution range of 1:500-1:1000, while immunohistochemistry applications use 1:20-1:200 . Researchers should validate these parameters in their specific experimental systems.

What are the most reliable positive controls for CASP10 antibody validation?

Reliable positive controls for CASP10 antibody validation include:

These validated controls help ensure antibody specificity and proper experimental setup . For optimal results in IHC applications, antigen retrieval is typically performed with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative option.

How can researchers differentiate between CASP10 isoforms in experimental systems?

Differentiating between CASP10 isoforms requires strategic experimental design due to their structural similarities. Western blot analysis can distinguish isoforms based on molecular weight: Pro-Caspase 10D appears at approximately 59 kDa, while Pro-Caspase 10A appears at 55 kDa . Their cleaved counterparts are detected at 47 kDa and 43 kDa, respectively.

Interestingly, expression patterns vary by cell type - B-lymphoblastoid cell lines (B-LCL) and SEE T blasts predominantly express uncleaved isoforms, while T-lymphoblastoid cell lines (T-LCL) display primarily cleaved forms . For comprehensive isoform analysis, researchers should use antibodies targeting conserved epitopes combined with size-based discrimination and potentially supplement with RT-PCR to confirm isoform-specific mRNA expression.

What methodological considerations should be made when assessing CASP10 in apoptosis research?

When investigating CASP10 in apoptosis research, several methodological considerations are critical:

• Temporal dynamics: Since caspase activation occurs in a time-dependent cascade, time-course experiments are essential to capture the sequential activation pattern.

• Cell-type specific expression: Expression patterns differ significantly between cell types. B-LCL and T blasts predominantly express uncleaved isoforms (Pro-Caspase 10D at 59 kDa and Pro-Caspase 10A at 55 kDa), while T-LCL predominantly display cleaved forms (47 kDa and 43 kDa) .

• Apoptosis induction method: Different apoptotic stimuli (FAS-mediated, TNF-α, granzyme B) may engage CASP10 through distinct mechanisms. For FAS-mediated apoptosis studies, parallel analysis of FAS (CD95) expression is advisable to exclude expression-level confounding factors .

• Complementary assays: Combine protein expression analysis (Western blot) with functional assays (caspase activity, apoptosis quantification) for a comprehensive assessment of CASP10's role.

How should researchers interpret conflicting data between CASP10 protein expression and functional assays?

Conflicting data between CASP10 protein expression and functional assays presents an interpretative challenge that requires careful analysis. Recent studies demonstrate this complexity - homozygous p.C401LfsX15 CASP10 variant carriers exhibit complete absence of CASP10 protein expression yet maintain normal FAS-mediated apoptosis function . This contradiction challenges the presumed essential role of CASP10 in apoptotic pathways.

When facing such contradictions, researchers should:

• Validate antibody specificity: Confirm that reduced signal represents actual protein reduction rather than epitope masking due to conformational changes or post-translational modifications.

• Assess RNA stability: Evaluate whether protein expression changes stem from transcriptional alterations using qRT-PCR, as demonstrated in studies showing that absent CASP10 protein corresponded with dramatically reduced RNA levels .

• Investigate compensatory mechanisms: Examine expression and activity of functionally related proteins (e.g., other caspases) that might compensate for CASP10 deficiency.

• Consider pathway redundancy: Interpret results in light of known pathway redundancies, particularly in the apoptotic cascade where multiple initiator caspases may fulfill similar functions.

What are the optimal experimental conditions for detecting CASP10 activation in different cell types?

Detecting CASP10 activation requires optimization based on cell type, as expression patterns vary significantly. The following table outlines optimal conditions for different experimental systems:

For activation studies, researchers should induce apoptosis using relevant stimuli (FAS ligand, staurosporine) and monitor the conversion from pro-forms to cleaved forms over a time course .

What is the current understanding of CASP10 variants in autoimmune lymphoproliferative syndrome (ALPS)?

Current evidence suggests:

• Common missense variants (p.V410I and p.Y446C) do not disrupt CASP10 expression or FAS-mediated apoptosis and should be considered polymorphisms rather than pathogenic mutations .

• Even the functionally significant variant p.C401LfsX15, which abolishes CASP10 expression, does not impair FAS-mediated apoptosis .

• ALPS patients without classical mutations in FAS, FASL, or CASP10 (designated ALPS-U) display a more complex phenotype characterized by multiorgan involvement and positive autoimmune markers compared to patients with mutations in these genes .

These findings suggest that CASP10 defects unlikely contribute significantly to ALPS pathogenesis since they don't impair FAS-mediated apoptosis, which is considered central to ALPS development .

How can researchers distinguish between pathogenic CASP10 variants and non-pathogenic polymorphisms?

Distinguishing between pathogenic CASP10 variants and non-pathogenic polymorphisms requires a multi-faceted approach integrating population data, functional assays, and clinical correlation:

• Population frequency analysis: High-frequency variants in healthy populations, like p.V410I and p.Y446C, are more likely polymorphisms than disease-causing mutations .

• Protein expression assessment: Western blot analysis can determine if variants affect protein expression levels. The p.C401LfsX15 variant abolishes protein expression, while missense variants like p.V410I and p.Y446C maintain normal expression .

• Functional apoptosis assays: Testing FAS-mediated apoptosis in cells expressing variant CASP10 provides critical functional evidence. Even variants that abolish CASP10 expression (p.C401LfsX15) may not impair apoptosis, challenging their pathogenic classification .

• Domain localization: Variants affecting the catalytic domain (like p.C401LfsX15 involving the QACQG catalytic site) are more likely to be functionally significant than those outside critical domains .

• RNA stability analysis: qRT-PCR can determine if protein expression changes stem from RNA instability, providing mechanistic insight into variant effects .

What methodologies are most effective for studying CASP10's role in primary immunodeficiencies?

Studying CASP10's role in primary immunodeficiencies requires a comprehensive methodological approach:

• Genetic screening: Next-generation sequencing panels targeting CASP10 alongside other apoptosis-related genes (FAS, FASL) provide the foundation for variant identification .

• Protein expression analysis: Western blotting with isoform-specific antibodies can detect alterations in CASP10 expression levels and processing patterns. This approach revealed that the p.C401LfsX15 variant abolishes protein expression while missense variants maintain normal levels .

• Functional apoptosis assays: Measuring FAS-mediated apoptosis in patient-derived T blasts provides critical functional data. This approach demonstrated that even CASP10-deficient cells maintain normal apoptotic responses, challenging the protein's presumed essential role .

• Surface marker analysis: Flow cytometry to quantify TCRαβ+CD4-CD8- (double-negative T cells) is particularly valuable, as increased levels of these cells are characteristic of ALPS patients .

• Biomarker assessment: Measuring elevated IL-10, IL-18, vitamin B12, and soluble FAS ligand levels provides supportive diagnostic information, particularly when genetic findings are ambiguous .

• Cell line models: Generating immortalized B-LCL and T-LCL from patient samples enables long-term mechanistic studies beyond the limitations of primary cell availability .

What are the optimal protocols for using CASP10 antibodies in Western blot applications?

For optimal Western blot detection of CASP10, researchers should follow these methodological guidelines:

• Sample preparation:

  • For apoptosis studies, include both untreated and apoptosis-induced samples (staurosporine-treated Jurkat cells or apoptosis-induced HeLa cells serve as positive controls)

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors to prevent degradation

• Antibody dilution and incubation:

  • Use a dilution range of 1:500-1:1000 for primary antibody

  • Incubate overnight at 4°C for optimal binding

  • HRP-conjugated secondary antibodies should be used at manufacturer-recommended dilutions

• Isoform detection considerations:

  • Be aware that different cell types express different isoforms predominantly

  • B-LCL and SEE T blasts mainly express uncleaved Pro-Caspase 10D (59 kDa) and Pro-Caspase 10A (55 kDa)

  • T-LCL primarily display cleaved forms (47 kDa and 43 kDa)

• Controls:

  • Include positive controls (staurosporine-treated Jurkat cells or apoptotic HeLa cells)

  • A loading control (β-actin, GAPDH) is essential for normalization

• Special considerations:

  • For variant studies, include samples from individuals with known CASP10 variants as references

  • When studying apoptosis, include time-course analysis to capture processing dynamics

How should researchers optimize CASP10 antibody-based immunofluorescence/immunocytochemistry protocols?

Optimizing CASP10 antibody-based immunofluorescence and immunocytochemistry requires attention to several technical parameters:

• Fixation method:

  • Paraformaldehyde (4%) is generally recommended for preserving protein epitopes

  • Methanol fixation may be preferred for detecting certain CASP10 epitopes

  • Compare multiple fixation methods to determine optimal conditions for your specific antibody

• Antibody dilution and incubation:

  • Use a dilution range of 1:20-1:200 for primary antibody

  • Overnight incubation at 4°C often yields optimal signal-to-noise ratio

  • Titrate antibody concentration in your specific cell system

• Cell-type considerations:

  • HepG2 cells have been validated for IF/ICC applications with CASP10 antibodies

  • Different cell types may require optimization of permeabilization conditions

• Controls and validation:

  • Include apoptosis-induced cells as positive controls

  • Perform peptide competition assays to confirm specificity

  • Consider dual staining with other apoptotic markers for colocalization studies

• Signal amplification:

  • For low-expression scenarios, consider tyramide signal amplification

  • Confocal microscopy provides better resolution for subcellular localization studies

What are the critical considerations for immunohistochemical detection of CASP10 in tissue samples?

Immunohistochemical detection of CASP10 in tissue samples presents unique challenges requiring specific methodological approaches:

• Antigen retrieval optimization:

  • TE buffer at pH 9.0 is the recommended primary method

  • Citrate buffer at pH 6.0 serves as an alternative approach

  • Optimization for specific tissues may be necessary

• Antibody dilution and detection:

  • Use a dilution range of 1:20-1:200 for primary antibody

  • Incubation times and temperatures should be optimized (typically overnight at 4°C)

  • DAB or AEC chromogens are commonly used for visualization

• Tissue-specific considerations:

  • Human endometrial cancer tissue has been validated as a positive control

  • Normal lymphoid tissues can serve as physiological reference standards

  • Tissues from ALPS patients may show altered expression patterns

• Controls:

  • Include known positive tissues (endometrial cancer)

  • Use appropriate negative controls (primary antibody omission, isotype controls)

  • Consider dual staining with cell-type specific markers to identify expressing populations

• Interpretation challenges:

  • Differentiate between specific staining and background

  • Consider counter-staining to provide tissue context

  • Quantification should include intensity scoring and percentage of positive cells

What are the best approaches for validating CASP10 antibody specificity in experimental systems?

Validating CASP10 antibody specificity is critical for experimental reliability. Researchers should implement these validation strategies:

• Genetic controls:

  • Samples from individuals with homozygous p.C401LfsX15 CASP10 variant provide excellent negative controls as they lack CASP10 protein expression

  • CRISPR/Cas9 knockout cell lines offer controlled validation systems

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide to verify signal specificity

  • Signal reduction or elimination confirms epitope-specific binding

• Multiple antibody validation:

  • Use antibodies recognizing different CASP10 epitopes to confirm expression patterns

  • Consistent detection across antibodies strengthens specificity claims

• Expression correlation:

  • Correlate protein detection with mRNA levels via qRT-PCR

  • Discrepancies may indicate antibody cross-reactivity or post-transcriptional regulation

• Isoform-specific considerations:

  • Verify detection of appropriate molecular weight bands corresponding to known isoforms

  • B-LCL and SEE T blasts express primarily uncleaved forms (59 kDa, 55 kDa)

  • T-LCL typically express cleaved forms (47 kDa, 43 kDa)

• Application-specific controls:

  • For WB: Include reducing and non-reducing conditions

  • For IF/ICC: Include peptide competition and secondary-only controls

  • For IHC: Include isotype controls and antibody titration

How does the role of CASP10 differ between cell types and developmental stages?

CASP10 exhibits significant cell-type specific expression patterns and functional roles:

• Lymphoid cell type variations:

  • B-lymphoblastoid cell lines (B-LCL) and SEE T blasts predominantly express uncleaved Pro-Caspase 10D (59 kDa) and Pro-Caspase 10A (55 kDa)

  • T-lymphoblastoid cell lines (T-LCL) mainly display cleaved forms (47 kDa and 43 kDa)

  • These differences likely reflect distinct regulatory mechanisms and functional requirements in different lymphocyte populations

• Isoform-specific functions:

  • Isoform 7 enhances NF-κB activity but induces only slight apoptosis

  • Isoform C is proteolytically inactive

  • This functional diversity suggests specialized roles beyond simple apoptosis execution

• Developmental context:

  • CASP10's role in lymphocyte homeostasis makes it particularly relevant during immune system development

  • The connection between CASP10 variants and autoimmune lymphoproliferative syndrome suggests developmental context-dependent functions

• Functional redundancy:

  • The finding that CASP10-deficient cells maintain normal FAS-mediated apoptosis suggests cell-type specific redundancy mechanisms

  • This redundancy may be developmentally regulated and tissue-specific

What emerging methodologies are advancing CASP10 research beyond traditional antibody applications?

CASP10 research is being transformed by several emerging methodologies that complement traditional antibody applications:

• CRISPR/Cas9 gene editing:

  • Precise knockout models enable functional validation of CASP10's role in apoptotic pathways

  • Introduction of specific variants allows direct assessment of their functional impact

  • Domain-specific mutations help map structure-function relationships

• Live-cell imaging with fluorescent reporters:

  • FRET-based caspase activity reporters enable real-time monitoring of CASP10 activation

  • This approach captures temporal dynamics lost in static antibody-based methods

  • Single-cell resolution reveals population heterogeneity in apoptotic responses

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) identify CASP10 interaction partners

  • Global proteome changes following CASP10 activation map its downstream effects

  • Post-translational modification analysis reveals regulatory mechanisms

• Single-cell technologies:

  • scRNA-seq captures transcriptional heterogeneity in CASP10 expression

  • CyTOF enables high-dimensional analysis of CASP10 in complex cell populations

  • These approaches reveal previously unappreciated cellular subsets with distinct CASP10 functionality

• Patient-derived organoids:

  • 3D culture systems from patients with CASP10 variants provide physiologically relevant models

  • These systems better recapitulate tissue-specific apoptotic regulation than traditional 2D cultures

What are the key considerations for researchers designing CASP10 antibody-based experiments?

When designing CASP10 antibody-based experiments, researchers should prioritize several critical considerations to ensure reliable and interpretable results:

• Antibody selection and validation:

  • Verify antibody specificity using appropriate controls, including samples known to lack CASP10 expression (e.g., cells with homozygous p.C401LfsX15 variant)

  • Select antibodies targeting epitopes preserved across relevant isoforms

  • Consider using multiple antibodies targeting different epitopes to confirm findings

• Experimental design optimization:

  • Include appropriate positive controls (staurosporine-treated Jurkat cells, apoptotic HeLa cells)

  • Optimize protocols for specific applications with recommended dilutions (WB: 1:500-1:1000; IHC: 1:20-1:200; IF/ICC: 1:20-1:200)

  • Account for cell-type specific expression patterns when interpreting results

• Functional context integration:

  • Complement expression analysis with functional apoptosis assays

  • Consider FAS-mediated apoptosis assessment alongside CASP10 expression studies

  • Incorporate temporal dynamics through time-course experiments

• Variant interpretation caution:

  • Exercise caution when attributing pathogenicity to CASP10 variants

  • Common variants (p.V410I, p.Y446C) appear to be non-pathogenic polymorphisms

  • Even functionally significant variants (p.C401LfsX15) may not impair apoptosis

By integrating these considerations, researchers can design robust experiments that advance understanding of CASP10's complex roles in apoptotic pathways and disease contexts.

How might future research resolve current contradictions in understanding CASP10's role in apoptosis and disease?

Resolving current contradictions in CASP10 research will require multifaceted approaches integrating advanced technologies and comprehensive experimental designs:

• Comprehensive variant functional characterization:

  • Systematic analysis of all reported CASP10 variants using consistent methodologies

  • Creation of isogenic cell lines with specific variants using CRISPR/Cas9 gene editing

  • Development of standardized functional assays for variant classification

• Investigation of compensatory mechanisms:

  • Identification of proteins that functionally compensate for CASP10 deficiency

  • Analysis of expression changes in other caspases in CASP10-deficient systems

  • Exploration of non-canonical pathways that might bypass CASP10 requirement

• Context-specific function mapping:

  • Tissue-specific and developmental stage-specific knockout models

  • Single-cell analysis to identify cellular subsets with distinct CASP10 dependencies

  • Stress condition analysis to identify scenarios where CASP10 becomes non-redundant

• Integration of clinical and molecular data:

  • Longitudinal studies of individuals with CASP10 variants to clarify disease associations

  • Correlation of molecular phenotypes with clinical manifestations

  • Multi-omics analysis of patient samples to identify biomarkers and molecular signatures

• Alternative function exploration:

  • Investigation of CASP10's potential non-apoptotic functions

  • Analysis of isoform-specific activities (e.g., isoform 7's role in NF-κB signaling)

  • Exploration of CASP10's role in inflammatory pathways beyond classical apoptosis

These approaches will help resolve the apparent contradiction between CASP10 deficiency and normal apoptotic function, providing clearer understanding of its true biological significance.