CARD16 Antibody

What is CARD16 and what cellular functions does it regulate?

CARD16, or caspase recruitment domain family member 16, is a protein with 197 amino acid residues and a molecular mass of 22.6 kDa in humans. It is widely expressed across many tissue types and plays important roles in the cellular response to hypoxia and the cellular response to lipopolysaccharides. The protein contains a caspase recruitment domain which is involved in protein-protein interactions during apoptosis and inflammation signaling pathways. CARD16 also serves as a marker for identifying Naive Regulatory T Cells, suggesting its importance in immune regulation . Understanding CARD16's function is crucial for designing experiments targeting inflammatory and immune response pathways.

How many isoforms of CARD16 exist and how do they differ?

Up to 2 different isoforms have been reported for the CARD16 protein in humans . These isoforms likely result from alternative splicing of the CARD16 gene, which can produce proteins with slightly different structures and potentially different functional properties. When designing experiments involving CARD16 detection, researchers should consider which isoform(s) they intend to target and select antibodies that can recognize the specific regions present in their isoform of interest. The differences between these isoforms may include variations in protein domain structure, post-translational modifications, or subcellular localization, which can affect antibody binding and experimental outcomes.

What are the common synonyms and alternative names for CARD16?

When searching literature and antibody resources, researchers should be aware that CARD16 is known by several alternative names. These synonyms include: CARD only domain-containing protein 1, CARD only protein, CARD-only protein 1, caspase recruitment domain-only protein 1, and caspase recruitment domain-containing protein 16 . Using all these terms in literature searches will ensure comprehensive coverage of available research. When ordering antibodies or designing experiments, cross-referencing these alternative names can help identify additional resources and prevent overlooking relevant information in databases and publications.

What are the most effective applications for CARD16 antibody detection?

Based on the available data, Western Blot is the most widely used application for CARD16 antibodies, followed by ELISA . When designing experiments, researchers should consider the specific requirements of each application:

The choice of application should be guided by the specific research question. For studying CARD16 expression levels, Western Blot provides semi-quantitative data with size verification. For precise quantification of CARD16 levels in biological fluids or cell lysates, ELISA would be more appropriate .

How should I validate a CARD16 antibody before using it in critical experiments?

Antibody validation is essential for ensuring experimental reproducibility. Based on the enhanced validation principles, researchers should employ at least two of the following five validation pillars for CARD16 antibodies :

• Orthogonal validation: Compare protein abundance levels determined by antibody-dependent methods (e.g., Western blot) with those from antibody-independent methods (e.g., mass spectrometry) across multiple cell lines. Correlation between methods increases confidence in antibody specificity .

• Genetic knockdown: Use siRNA or CRISPR to reduce CARD16 expression and confirm corresponding reduction in antibody signal. This confirms the antibody is detecting the intended target .

• Recombinant expression: Overexpress CARD16 in a cell line with low endogenous expression and verify increased antibody signal .

• Independent antibodies: Use multiple antibodies targeting different epitopes of CARD16 and compare their staining patterns. Concordant results increase confidence in specificity .

• Capture mass spectrometry: Cut out Western blot bands at the apparent size detected by the antibody and perform mass spectrometry to confirm the presence of CARD16 peptides .

For optimal validation, combine at least two methods, preferably including genetic knockdown or recombinant expression approaches.

What cell lines are recommended for CARD16 antibody testing and validation?

Based on established validation protocols, a panel of diverse cell lines should be used to test CARD16 antibody specificity and sensitivity . While specific cell lines for CARD16 expression are not explicitly mentioned in the search results, the general validation approach suggests using:

• Cell lines with known high CARD16 expression (positive controls)

• Cell lines with low or no CARD16 expression (negative controls)

• A panel of 4-8 different cell lines for orthogonal validation

Using standardized cell line panels allows for reproducible validation across different laboratories. When selecting cell lines, consider tissue relevance to your research question and availability of transcriptomic data to confirm CARD16 expression levels before antibody testing. Cell lines commonly used for immune-related protein validation include THP-1, Jurkat, and primary human PBMCs .

How can I distinguish between CARD16 isoforms in Western blot analysis?

Distinguishing between CARD16 isoforms requires careful experimental design and data analysis:

• Molecular weight analysis: The canonical CARD16 protein has a reported mass of 22.6 kDa . Different isoforms may show slight variations in migration on SDS-PAGE gels. Always include molecular weight markers and run positive controls with known isoform expression.

• Isoform-specific antibodies: Select antibodies raised against epitopes unique to specific isoforms when possible. Check the antibody datasheet for information on which isoforms the antibody can detect.

• 2D gel electrophoresis: For challenging cases, combine isoelectric focusing with SDS-PAGE to separate isoforms based on both charge and size.

• Mass spectrometry validation: Use capture MS to identify peptides specific to each isoform in gel bands of interest .

When reporting results, clearly specify which isoform(s) your antibody detects and include this information in publications to improve reproducibility.

What controls are essential when performing Western blot with CARD16 antibodies?

Proper controls are critical for reliable interpretation of CARD16 Western blot results:

• Positive control: Include a sample known to express CARD16 (e.g., recombinant CARD16 protein or lysate from cells with confirmed expression).

• Negative control: Include samples from genetic knockdown experiments or tissues/cells known not to express CARD16.

• Loading control: Use antibodies against housekeeping proteins (e.g., GAPDH, β-actin) to ensure equal loading across lanes.

• Primary antibody control: Perform a blot without primary antibody to identify non-specific binding from secondary antibodies.

• Competitive peptide blocking: Pre-incubate the antibody with excess target peptide to confirm binding specificity.

For enhanced validation, genetic knockdown controls provide compelling evidence of antibody specificity. The signal reduction should correlate with the knockdown efficiency as measured by qPCR .

How does CARD16 antibody cross-reactivity with other CARD-domain proteins impact experimental interpretation?

Cross-reactivity with other CARD-domain proteins is a significant concern when working with CARD16 antibodies:

• Sequence homology: CARD16 shares structural similarities with other CARD-domain proteins, potentially leading to cross-reactivity. Cross-reactivity is more likely with polyclonal antibodies that recognize multiple epitopes.

• Validation requirements: To assess potential cross-reactivity, perform:

  • Western blots in cells expressing related CARD proteins but lacking CARD16

  • Immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody

  • Competition assays with recombinant related CARD proteins

• Data interpretation: When interpreting results, consider:

  • The precise molecular weight of observed bands (CARD16 is 22.6 kDa)

  • Expression patterns across tissues/cells compared to known CARD16 distribution

  • Results from orthogonal methods that don't rely on antibody specificity

To minimize cross-reactivity issues, preferentially use monoclonal antibodies targeting unique epitopes of CARD16 that are absent in related proteins.

How can CARD16 antibodies be utilized in studying regulatory T cell populations?

Since CARD16 serves as a marker for Naive Regulatory T Cells , researchers can leverage CARD16 antibodies for advanced T cell studies:

• Flow cytometry applications:

  • Use fluorophore-conjugated CARD16 antibodies in multi-parameter flow panels

  • Combine with other Treg markers (CD4, CD25, FOXP3) for comprehensive phenotyping

  • Sort CARD16+ populations for functional studies or transcriptional profiling

• Single-cell analysis:

  • Incorporate CARD16 antibodies in CyTOF/mass cytometry panels for high-dimensional analysis

  • Use in single-cell Western blot systems for protein quantification at the single-cell level

• Functional correlation:

  • Assess CARD16 expression changes during Treg activation or differentiation

  • Correlate CARD16 levels with suppressive capacity using in vitro suppression assays

When designing such experiments, confirm antibody compatibility with your specific application through pilot studies and titration experiments to determine optimal concentrations.

Can CARD16 antibodies be adapted for CAR-T cell therapeutic research?

While CARD16 itself is not mentioned in CAR-T cell research in the provided search results, the principles of CD16-based chimeric antigen receptors (CARs) can inform potential applications for CARD16-related research:

• CARD16 as a potential CAR target: If CARD16 shows restricted expression in certain cancer types, antibodies against CARD16 could potentially be adapted for CAR-T cell development, similar to other targeted therapies .

• Methodological considerations:

  • Ensure extremely high antibody specificity through multiple validation methods

  • Test antibody binding to CARD16 in native conformations on cell surfaces

  • Validate absence of binding to healthy tissues to prevent off-target effects

• Adaptation process:

  • Engineer single-chain variable fragments (scFvs) from validated CARD16 antibodies

  • Test binding affinity and specificity of the scFv constructs

  • Incorporate into standard CAR constructs with appropriate co-stimulatory domains

For such advanced applications, researchers should first establish CARD16's expression pattern in relevant cancer types compared to healthy tissues and validate antibody specificity through multiple independent methods .

What approaches enable studying CARD16's role in hypoxia response pathways?

CARD16 is involved in cellular responses to hypoxia , and specialized antibody applications can help elucidate its specific functions:

• Time-course experiments:

  • Culture cells under controlled hypoxic conditions (1-5% O₂)

  • Harvest at multiple time points (0, 2, 6, 12, 24, 48 hours)

  • Perform Western blot with validated CARD16 antibodies to track expression changes

  • Correlate with HIF-1α levels as a positive control for hypoxia induction

• Co-immunoprecipitation studies:

  • Use validated CARD16 antibodies to pull down protein complexes

  • Identify interaction partners under normoxic vs. hypoxic conditions

  • Confirm specific interactions using reverse co-IP and proximity ligation assays

• Subcellular localization changes:

  • Perform immunofluorescence with CARD16 antibodies in cells under normoxia vs. hypoxia

  • Co-stain with organelle markers to track potential translocation events

  • Quantify changes in localization patterns using high-content imaging

When designing these experiments, include appropriate controls for antibody specificity and ensure consistent hypoxic conditions using calibrated equipment.

What are common sources of false positive signals when using CARD16 antibodies?

• Cross-reactivity: CARD16 antibodies may bind to other CARD-domain proteins due to structural similarities. Validate specificity using knockout/knockdown controls .

• Non-specific binding: Secondary antibodies may bind to endogenous Fc receptors in certain cell types. Use appropriate blocking reagents and include secondary-only controls.

• Inadequate validation: Using antibodies that haven't undergone rigorous validation can lead to misleading results. Apply multiple validation strategies as described in the five-pillar approach .

• Sample preparation issues: Overfixation can create artifactual staining patterns. Optimize fixation protocols for each application and include appropriate technical controls.

• Concentration-dependent non-specificity: Excessive antibody concentrations can increase background and non-specific binding. Perform titration experiments to determine optimal concentration.

To minimize false positives, always include negative controls and validate antibodies using at least two independent methods from the five validation pillars .

How should CARD16 antibody validation data be documented and reported in publications?

Proper documentation of antibody validation is essential for research reproducibility:

• Minimal reporting requirements:

  • Complete antibody information (supplier, catalog number, lot number, RRID)

  • Validation methods employed (from the five pillars)

  • Experimental conditions (dilution, incubation time, temperature)

  • All controls used (positive, negative, loading)

• Recommended additional documentation:

  • Images of full unedited blots including molecular weight markers

  • Quantification methods and software used

  • Antibody titration data demonstrating optimal concentration

  • Links to repositories containing validation data

• Structured reporting format:

  • Include a dedicated antibody validation section in methods

  • Provide a supplementary table with all antibody details

  • Deposit raw validation data in appropriate repositories

  • Reference previous validation studies for the same antibody

Following structured reporting guidelines increases transparency and allows other researchers to evaluate and reproduce findings. When possible, cite resources like the Human Protein Atlas (www.proteinatlas.org) where antibody validation data is publicly available .

What strategies can overcome weak or inconsistent CARD16 antibody signals?

When facing weak or inconsistent signals with CARD16 antibodies, consider these methodological approaches:

• Sample enrichment strategies:

  • Immunoprecipitate CARD16 before Western blotting to concentrate the target

  • Use subcellular fractionation to enrich compartments where CARD16 is localized

  • Induce CARD16 expression through appropriate stimuli (e.g., hypoxia or LPS treatment)

• Signal amplification methods:

  • Employ tyramide signal amplification for immunohistochemistry/immunofluorescence

  • Use high-sensitivity ECL substrates for Western blot

  • Consider biotin-streptavidin amplification systems

• Antibody optimization:

  • Test multiple antibodies targeting different epitopes of CARD16

  • Optimize antibody concentration through careful titration experiments

  • Adjust incubation conditions (time, temperature, buffer composition)

• Protocol modifications:

  • Test different antigen retrieval methods for fixed samples

  • Optimize blocking conditions to improve signal-to-noise ratio

  • Explore alternative detection systems (fluorescent vs. chromogenic)

For particularly challenging samples, consider combining antibody-based detection with mass spectrometry validation to confirm protein identity .