CARD19 Antibody

What is CARD19 and why is it important to study?

CARD19 is a mitochondrial membrane protein containing an N-terminal caspase activation and recruitment domain (CARD) and a C-terminal transmembrane domain . It functions as a critical regulator in cellular processes including pyroptosis and inflammatory signaling pathways. The importance of studying CARD19 stems from its roles in:

• Regulating cell death mechanisms, particularly pyroptosis, which is crucial in inflammatory responses

• Modulating TAK1 activation through interaction with TAB2, affecting NF-κB signaling pathways

• Influencing B-cell tolerance mechanisms and potentially autoimmunity development

• Contributing to mitochondria-associated immune signaling, similar to other mitochondrial CARD-containing proteins like MAVS

Understanding CARD19 function provides valuable insights into fundamental immunological processes and potential therapeutic targets for inflammatory and autoimmune conditions.

How are CARD19 antibodies typically used in basic research applications?

CARD19 antibodies serve multiple fundamental research purposes:

• Protein detection: Western blotting techniques utilize CARD19 antibodies to identify and quantify protein expression levels in cell lysates. Researchers commonly employ polyclonal antibodies targeting the N-terminal 100 amino acids of mouse CARD19 for immunoblotting experiments .

• Localization studies: Immunocytochemistry (ICC) and immunohistochemistry (IHC) applications help visualize CARD19's subcellular localization, particularly its association with mitochondrial membranes.

• Protein interaction analyses: Co-immunoprecipitation experiments using CARD19 antibodies have revealed important protein-protein interactions, such as the CARD19-TAK1 interaction that influences NF-κB signaling pathways .

• Functional validation: CARD19 antibodies help validate knockout or knockdown models by confirming the absence or reduction of CARD19 protein expression, as demonstrated in studies with CARD19-deficient mice .

These applications establish foundational knowledge about CARD19's expression patterns, localization, and interaction partners.

What are the key considerations for selecting a CARD19 antibody for research?

When selecting a CARD19 antibody, researchers should consider several critical factors:

• Antibody specificity: Validate that the antibody recognizes CARD19 specifically without cross-reactivity to other CARD-containing proteins. Cross-validation with multiple antibodies targeting different epitopes or use of CARD19-deficient controls can confirm specificity .

• Species reactivity: Ensure the antibody recognizes CARD19 from your experimental species. Many commercially available antibodies target human or mouse CARD19, but cross-reactivity between species may vary.

• Application compatibility: Verify the antibody is validated for your specific application (Western blot, ICC/IHC, flow cytometry, etc.). Some antibodies perform well in denatured protein detection (Western blot) but poorly in detecting native conformations (immunoprecipitation).

• Epitope location: Consider whether targeting the N-terminal CARD domain or C-terminal transmembrane domain aligns with your research questions. For instance, polyclonal antibodies targeting the N-terminal 100 amino acids have been successfully used in immunoblotting studies .

• Clonality: Polyclonal antibodies offer broader epitope recognition but potential batch-to-batch variability, while monoclonal antibodies provide consistent specificity for a single epitope.

Thorough antibody validation using appropriate positive and negative controls remains essential regardless of manufacturer specifications.

How can CARD19 antibodies be used to investigate pyroptosis mechanisms?

CARD19 antibodies provide sophisticated tools for dissecting pyroptosis regulation through multiple experimental approaches:

• Temporal analysis of CARD19 localization: Using immunofluorescence with CARD19 antibodies, researchers can track protein redistribution during pyroptosis induction. This reveals whether CARD19 translocates from mitochondria during inflammatory activation.

• Co-localization with pyroptotic machinery: Dual-labeling experiments with CARD19 antibodies and markers for inflammasomes (NLRP3, ASC), gasdermin D, or caspase-1 can elucidate spatial relationships during pyroptosis progression. Research indicates CARD19 affects caspase-1 processing and GSDMD cleavage dynamics during Salmonella Typhimurium infection .

• Protein complex analysis: Proximity ligation assays using CARD19 antibodies can identify transient protein-protein interactions occurring during pyroptosis activation that might be missed by conventional co-immunoprecipitation.

• Phosphorylation-specific detection: Phospho-specific CARD19 antibodies could potentially identify post-translational modifications regulating CARD19 function during pyroptotic signaling, though these specialized tools may require custom development.

Experimental data indicates that CARD19-deficient macrophages display reduced levels of cell death following bacterial infection, with equivalent initial caspase-1 processing but altered release kinetics of cleaved caspase-1 into cell supernatants . This suggests CARD19 functions downstream of inflammasome assembly and initial caspase activation but upstream of terminal cellular lysis.

What methodological approaches can resolve contradictory findings in CARD19 antibody experiments?

Researchers encountering conflicting results with CARD19 antibodies should implement systematic troubleshooting strategies:

• Antibody validation pipeline:

  • Test multiple CARD19 antibodies targeting different epitopes

  • Include CARD19-deficient samples as negative controls

  • Perform peptide competition assays to confirm binding specificity

  • Verify antibody performance across multiple experimental conditions

• Cell type and context considerations: CARD19 function appears cell-type dependent, with distinct phenotypes observed between thioglycolate-elicited peritoneal macrophages and resident peritoneal macrophages . When contradictory findings emerge, compare:

  • Cell type-specific CARD19 expression levels

  • Activation state differences (resting vs. stimulated)

  • Cell isolation and culture conditions

  • Genetic background effects

• Technical variables standardization:

  • Fixation methods (critical for preserving mitochondrial structures)

  • Antibody concentrations and incubation parameters

  • Detection systems (fluorescence vs. chemiluminescence)

  • Quantification methodologies and statistical approaches

• Independent validation techniques: Complement antibody-based detection with orthogonal methods:

  • mRNA expression analysis (RT-qPCR, RNA-seq)

  • Tagged CARD19 expression constructs

  • Mass spectrometry-based protein identification

These systematic approaches can help reconcile apparently contradictory findings by identifying underlying methodological variables or genuine biological differences.

How can researchers use CARD19 antibodies to study its role in NF-κB signaling pathways?

CARD19's interaction with the TAK1/NF-κB pathway offers a complex experimental landscape requiring sophisticated antibody applications:

• Signaling complex immunoprecipitation: CARD19 antibodies can immunoprecipitate protein complexes to study dynamic interactions during NF-κB activation. Research demonstrates that CARD19 strongly interacts with TAK1 but not other components of the CBM signalosome . Sequential immunoprecipitation experiments can reveal the composition and stoichiometry of these complexes.

• In vitro ubiquitination assays: Combining CARD19 antibodies with antibodies against TAK1, TAB2, and ubiquitin enables analysis of how CARD19 inhibits TAB2-mediated TAK1 ubiquitination . These assays can include:

  • Detection of TAK1 ubiquitination status

  • Identification of specific ubiquitin linkage types (K48 vs. K63)

  • Temporal dynamics of ubiquitination following stimulation

• Chromatin immunoprecipitation (ChIP) analysis: Using antibodies against NF-κB components after CARD19 manipulation helps identify genomic targets affected by CARD19-mediated NF-κB regulation. This approach can reveal which specific gene sets are most sensitive to CARD19's regulatory effects.

• Phospho-specific protein analysis: Antibodies against phosphorylated forms of IκB, IKK, and NF-κB subunits can track signaling cascade activation in CARD19-sufficient versus CARD19-deficient conditions. Research shows CARD19 deficiency enhances BCR/TAK1-mediated NF-κB activation with downstream consequences for B-cell tolerance .

These methodologies help dissect the molecular mechanisms through which CARD19 regulates TAK1 activation and subsequent NF-κB signaling, with particular relevance to B-cell self-tolerance and autoimmunity.

What are the optimal protocols for immunoblotting with CARD19 antibodies?

Successful immunoblotting for CARD19 requires attention to specific technical parameters:

Sample Preparation:

• Use RIPA buffer supplemented with protease inhibitors for efficient extraction of membrane-associated CARD19

• Include phosphatase inhibitors if investigating phosphorylated forms of CARD19

• For mitochondrial enrichment, consider subcellular fractionation techniques

• Determine optimal protein loading (typically 20-50 μg total protein) through titration experiments

Electrophoresis and Transfer:

• Resolve proteins on 10-12% SDS-PAGE gels for optimal separation

• Use PVDF membranes for better protein retention

• Transfer at lower voltage (30V) overnight at 4°C to ensure efficient transfer of transmembrane proteins

Antibody Incubation:

• Blocking with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary CARD19 antibody incubation at 1:1000 dilution overnight at 4°C

• Extended washing (4 × 10 minutes) to minimize background

• HRP-conjugated secondary antibody incubation at 1:5000 for 1 hour at room temperature

Detection Considerations:

• Enhanced chemiluminescence (ECL) detection with exposure times optimized for CARD19's expression level

• Consider staining for loading controls on separate membranes to avoid stripping

Research groups have successfully used rabbit polyclonal antibodies targeting the N-terminal 100 amino acids of mouse CARD19 in immunoblotting applications . For human CARD19 detection, validation of antibody specificity with appropriate positive and negative controls remains essential.

How should researchers design experiments to study CARD19 in different cellular contexts?

Experimental design for CARD19 research across cellular contexts requires systematic consideration of multiple variables:

Cell Type Selection:

• Macrophages: Distinct CARD19 functions are observed between bone marrow-derived macrophages (BMDMs) and peritoneal macrophages. Further differences exist between large and small peritoneal macrophage populations (LPM and SPM) .

• B cells: CARD19's role in B-cell tolerance makes primary B cells, particularly self-reactive models, valuable experimental systems .

• Cell lines: Useful for mechanism studies but require validation in primary cells.

Experimental Controls Matrix:

Context-Dependent Protocols:

• For pyroptosis studies: Standardize MOI and infection times when using bacterial stimuli like S. Typhimurium .

• For NF-κB signaling: Include both early (minutes) and late (hours) timepoints to capture immediate signaling events and downstream transcriptional responses .

• For autoimmunity models: Consider both in vitro B-cell activation models and in vivo systems like Bm12-induced experimental lupus erythematosus .

Researchers should systematically document cell isolation protocols, culture conditions, passage numbers, and stimulation parameters to enable reproducibility and meaningful cross-study comparisons.

What are the technical challenges in developing phospho-specific CARD19 antibodies?

Development of phospho-specific CARD19 antibodies presents several technical challenges that researchers must address:

Epitope Identification Challenges:

• Prediction of phosphorylation sites requires computational analysis with tools like NetPhos, combined with mass spectrometry validation

• Phosphorylation may be transient or occur at low stoichiometry, complicating detection

• Multiple phosphorylation sites may exist with different functional consequences

Antibody Production Strategies:

• Synthetic phosphopeptide approach:

  • Design phosphopeptides corresponding to predicted phosphorylation sites

  • Include carrier proteins (KLH or BSA) for immunization

  • Implement dual-purification strategy:

    • Positive selection on phosphopeptide columns

    • Negative selection on non-phosphorylated peptide columns

• Validation requirements:

  • Test antibody recognition with phosphatase-treated samples as negative controls

  • Validate with phosphomimetic and phosphodead CARD19 mutants

  • Confirm signaling context-appropriate recognition patterns

Context-Dependent Phosphorylation:

• CARD19 phosphorylation may differ between pyroptotic and NF-κB signaling contexts

• Stimulation conditions must be optimized to maximize phosphorylation for validation

• Cell type differences may necessitate separate validation in macrophages versus B cells

While no phospho-specific CARD19 antibodies are described in the provided research materials, their development would significantly advance understanding of CARD19 regulation, particularly given its interactions with signaling kinases like TAK1 .

How can researchers address non-specific binding in CARD19 immunodetection?

Non-specific binding represents a common challenge in CARD19 antibody applications that can be systematically addressed:

Sources of Non-Specificity:

• Cross-reactivity with other CARD-containing proteins

• Non-specific binding to mitochondrial components

• Secondary antibody background

• Inconsistent sample preparation

Optimization Strategies:

Validation Approaches:

• Perform peptide competition assays using the immunizing peptide

• Compare results from multiple CARD19 antibodies targeting different epitopes

• Include genetic controls (CARD19-knockout or knockdown samples)

• Implement orthogonal detection methods (mass spectrometry)

Researchers should document optimization efforts and implement standardized protocols once conditions are established to ensure consistent results across experiments.

What approach should be taken when CARD19 antibody results conflict with genetic knockout phenotypes?

Discrepancies between antibody-based results and genetic knockout phenotypes require systematic investigation:

Potential Sources of Discrepancy:

• Alternative splicing: CARD19 may exist in multiple isoforms not all affected by genetic manipulation

• Compensatory mechanisms: Long-term CARD19 deficiency may trigger upregulation of related proteins

• Dual functions: CARD19 may have scaffold-independent functions not detected by antibodies

• Technical artifacts: Antibody specificity issues or knockout inefficiency

Systematic Resolution Approach:

• Validate the genetic model:

  • Confirm complete CARD19 deletion using PCR across multiple exons

  • Verify absence of truncated proteins using multiple antibodies targeting different regions

  • Check for potential off-target effects through whole-genome sequencing

• Extend antibody validation:

  • Test multiple antibodies targeting different CARD19 epitopes

  • Include appropriate positive and negative controls

  • Validate antibody specificity through immunoprecipitation followed by mass spectrometry

• Implement complementary approaches:

  • Conduct rescue experiments with CARD19 re-expression in knockout cells

  • Use alternative gene silencing methods (siRNA, shRNA) to confirm phenotypes

  • Deploy domain-specific mutants to isolate function-specific effects

• Design time-course experiments:

  • Acute versus chronic CARD19 depletion may yield different results

  • Implement inducible knockout systems to distinguish immediate from adaptive responses

These approaches can help distinguish genuine biological complexity from technical artifacts when investigating CARD19 function.

How might computational approaches enhance CARD19 antibody design and application?

Computational methodologies offer promising avenues to improve CARD19 antibody development and experimental applications:

Epitope Prediction and Antibody Design:

• Machine learning algorithms can predict optimal CARD19 epitopes for antibody generation based on:

  • Protein surface accessibility

  • Sequence conservation across species

  • Structural distinctiveness from other CARD-containing proteins

  • Post-translational modification sites

• Generative antibody design platforms like GUIDE (Generative Unconstrained Intelligent Drug Engineering) could optimize CARD19 antibody sequences for:

  • Improved affinity to specific CARD19 regions

  • Reduced cross-reactivity with related proteins

  • Enhanced stability and performance across applications

Advanced Data Analysis for CARD19 Research:

• Machine learning analysis of immunofluorescence data can improve quantification of CARD19 co-localization with cellular structures

• Computational modeling of CARD19 signaling networks can predict key interaction points for antibody-based validation

• Trajectory analysis of antibody binding kinetics can improve experimental design for time-resolved studies

Integrative Research Applications:

• Systems biology approaches combining CARD19 antibody data with transcriptomics and proteomics can reveal broader pathway impacts

• Mathematical modeling of antibody kinetics, similar to approaches used in SARS-CoV-2 research, could predict optimal experimental time points for CARD19 detection

• Visualization algorithms can enhance detection of subtle CARD19 localization changes during cellular activation

These computational approaches could significantly accelerate CARD19 research by improving antibody design, optimizing experimental protocols, and enhancing data interpretation.

What are the emerging applications of CARD19 antibodies in autoimmunity research?

CARD19's role in B-cell tolerance and NF-κB regulation positions it as a compelling target for autoimmunity research, with several promising applications:

Biomarker Development:

• CARD19 antibodies could help identify altered CARD19 expression or regulation in autoimmune patient samples

• Monitoring CARD19 phosphorylation or complex formation might serve as biomarkers for NF-κB dysregulation in autoimmune conditions

• Single-cell analysis using CARD19 antibodies could identify cell subpopulations with altered signaling in autoimmune contexts

Mechanistic Investigations:

• CARD19 antibodies can help dissect the regulatory mechanisms controlling B-cell tolerance:

  • Track CARD19-TAK1 interactions during BCR activation in self-reactive B cells

  • Monitor downstream effects on Egr2/3 and c-Cbl/Cbl-b expression

  • Investigate CARD19's role in receptor editing and clonal deletion processes

Therapeutic Target Validation:

• CARD19 antibodies can assist in validating targeting strategies that might modulate autoimmune responses

• Monitoring CARD19 complex formation following experimental therapeutics treatment

• Identifying cell-type specific differences in CARD19 regulation that might inform precision medicine approaches

Animal Model Applications:

• CARD19 antibodies enable detailed phenotyping of experimental autoimmune models

• Research demonstrates CARD19 deficiency prevents Bm12-induced experimental systemic lupus erythematosus , suggesting monitoring CARD19 expression could help predict disease progression

• Combination with phospho-specific antibodies against NF-κB pathway components could reveal activation patterns in autoimmune settings

These applications position CARD19 antibodies as valuable tools for understanding fundamental autoimmune mechanisms and potentially developing novel therapeutic approaches.