CARD18 Antibody

What is CARD18 and what is its role in cellular signaling pathways?

CARD18 (also known as ICEBERG, pseudo-ICE, or UNQ5804) is a caspase recruitment domain-containing protein that functions as a caspase-1 inhibitor. It inhibits the generation of interleukin-1β (IL-1β) by directly interacting with caspase-1 and preventing its association with RIP2, thereby down-regulating the release of IL-1β . CARD18 contains a caspase recruitment domain (CARD) that facilitates this binding to caspase-1 . This inhibitory function positions CARD18 as a negative regulator of inflammatory processes mediated by the inflammasome pathway.

Structurally, CARD18 is a relatively small protein of 90 amino acids, consisting primarily of the CARD domain, which is crucial for protein-protein interactions in the inflammatory signaling cascade . The protein's regulatory role in inflammation makes it particularly relevant in research on inflammatory skin conditions and keratinocyte biology.

Where is CARD18 predominantly expressed and how is its expression regulated?

CARD18 exhibits highly tissue-specific expression patterns. Research has demonstrated that CARD18 mRNA is expressed in the epidermis at more than 100-fold higher levels compared to any other human tissue . Within the epidermis, CARD18 expression is further localized specifically to the granular layer .

The expression of CARD18 is significantly upregulated during terminal differentiation of keratinocytes at both mRNA and protein levels, suggesting it plays a specific role in this process . Additionally, environmental factors can modulate CARD18 expression:

• UVB irradiation of skin explants increases epidermal expression of CARD18, indicating a potential stress-response function

• In inflammatory skin conditions, CARD18 expression patterns are altered; notably, in lesional epidermis of patients with lichen planus, CARD18 expression is either greatly diminished or entirely absent, while remaining comparable to normal skin in non-lesional areas

These findings suggest that CARD18 expression is regulated by both differentiation-associated factors and external stressors, with potential implications for its role in inflammatory skin conditions.

What are the validated applications for CARD18 antibodies in research?

CARD18 antibodies have been validated for multiple research applications, with varying degrees of validation across different commercial sources. The most commonly validated applications include:

When selecting a CARD18 antibody for your research, it's advisable to choose antibodies that have been specifically validated for your application of interest and experimental system . The specificity and performance of antibodies can vary significantly between applications, so reviewing validation data is essential.

For optimal results in detection of native CARD18, antibodies raised against full-length protein or the functional CARD domain may provide better recognition compared to those targeting only small peptide sequences .

What are the recommended storage and handling conditions for CARD18 antibodies?

Proper storage and handling of CARD18 antibodies is crucial for maintaining their activity and specificity. Based on manufacturer recommendations across multiple sources:

• Storage temperature: Most CARD18 antibodies should be stored at -20°C or -80°C for long-term preservation

• Aliquoting: Upon receipt, it's recommended to prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Freeze-thaw limitations: Limit to 2-3 freeze-thaw cycles to preserve antibody functionality

• Working solution: For immunohistochemistry applications, typical dilutions range from 1:50 to 1:200, but optimal concentrations should be determined empirically for each experimental system

• Buffer composition: Most commercial CARD18 antibodies are supplied in buffered aqueous glycerol solutions containing preservatives such as 0.03% Proclin 300

When working with CARD18 antibodies for the first time, it's advisable to perform a dilution series to determine the optimal concentration for your specific application and experimental system.

What methodological approaches can be used to study CARD18's role in inflammatory skin diseases?

Investigating CARD18 in inflammatory skin conditions requires multiple complementary approaches:

• Comparative expression analysis:

  • Quantitative RT-PCR to measure CARD18 mRNA levels in healthy vs. diseased skin samples

  • Immunohistochemistry to visualize CARD18 protein localization in tissue sections

  • Western blot analysis to quantify protein expression levels

• Functional studies in keratinocyte models:

  • In vitro differentiation experiments using primary keratinocytes or HaCaT cells

  • siRNA-mediated knockdown of CARD18 expression to assess impact on differentiation markers and inflammatory cytokine production

  • UV irradiation studies to examine stress-induced regulation of CARD18

• Three-dimensional skin equivalents:

  • 3D skin equivalent cultures with modulated CARD18 expression

  • Analysis of stratum corneum formation and barrier function

  • Evaluation of inflammasome activation markers

Research has shown that CARD18 expression is dramatically altered in lichen planus lesions, suggesting it may play a role in disease pathogenesis . When designing experiments to investigate CARD18 in inflammatory conditions, researchers should consider including appropriate disease controls and examining interactions with other inflammasome components.

How can researchers validate the specificity of CARD18 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable research results. For CARD18 antibodies, consider implementing these validation strategies:

• Positive and negative controls:

  • Positive control: Use tissues with known high CARD18 expression (e.g., normal epidermis granular layer)

  • Negative control: Use tissues with minimal CARD18 expression or CARD18 knockout models

  • Transfected cell lysates: HEK-293T cells transfected with CARD18 expression vectors serve as excellent positive controls

• Knockdown validation:

  • Perform siRNA-mediated knockdown of CARD18 in appropriate cell models

  • Confirm signal reduction by Western blot or immunostaining

  • Studies have demonstrated successful CARD18 suppression in skin equivalent cultures using siRNAs

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide/protein

  • Observe elimination or reduction of specific signal

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of CARD18

  • Compare staining/blotting patterns across antibodies

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (~10 kDa for untagged CARD18)

  • For tagged recombinant proteins, account for the additional size of the tag

These validation steps should be conducted in the specific experimental system and application you intend to use, as antibody performance can vary across contexts.

What are the advantages and limitations of polyclonal versus monoclonal CARD18 antibodies?

Most commercially available CARD18 antibodies are polyclonal, typically raised in rabbit or mouse . Each antibody type offers distinct advantages and limitations for CARD18 research:

Polyclonal CARD18 Antibodies:

Advantages:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• More tolerant to minor changes in protein conformation or modifications

• Generally less expensive and easier to produce

• Available from multiple commercial sources with validation data

Limitations:

• Lot-to-lot variability can affect reproducibility

• May exhibit higher background in some applications

• Higher potential for cross-reactivity with related proteins

Monoclonal CARD18 Antibodies:

Advantages:

• Consistent performance across different lots

• Highly specific for a single epitope

• May provide cleaner results with less background

• Preferred for applications requiring high specificity

Limitations:

• Limited commercial availability for CARD18

• May have reduced sensitivity compared to polyclonal antibodies

• Could fail to detect CARD18 if the specific epitope is masked or modified

• May be more sensitive to fixation conditions in IHC applications

When selecting between polyclonal and monoclonal antibodies for CARD18 research, consider your specific application requirements, the need for reproducibility across experiments, and the available validation data for each antibody option.

How does CARD18 expression differ between normal and pathological skin conditions?

Research has revealed significant differences in CARD18 expression patterns between normal skin and various pathological conditions:

Normal Skin:

• CARD18 is highly expressed in the granular layer of the epidermis

• Expression increases during terminal differentiation of keratinocytes

• UVB exposure upregulates CARD18 expression, suggesting a role in stress response

Pathological Conditions:

• Lichen Planus: CARD18 expression is either greatly diminished or entirely absent in lesional epidermis, while expression in non-lesional areas remains comparable to normal skin

• Psoriasis: Research has not shown consistent regulation of CARD18 expression in psoriatic skin, suggesting disease-specific alterations

These differential expression patterns suggest CARD18 may play distinct roles in various inflammatory skin conditions. When designing studies to investigate these differences, researchers should consider:

• Analyzing paired samples (lesional vs. non-lesional) from the same patient

• Including multiple disease conditions for comparative analysis

• Correlating CARD18 expression with inflammation markers and disease severity

• Examining expression of other inflammasome components alongside CARD18

The disease-specific alterations in CARD18 expression highlight its potential role in the pathogenesis of certain inflammatory skin conditions and suggest it could serve as a biomarker or therapeutic target.

What techniques can be used to study CARD18 interactions with caspase-1 and other binding partners?

Studying CARD18's interactions with caspase-1 and other potential binding partners requires specialized techniques to capture protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CARD18 antibodies to pull down protein complexes

  • Analyze precipitated proteins by Western blot for caspase-1 and other suspected interaction partners

  • Reverse Co-IP (using anti-caspase-1 antibodies) can confirm interactions

• Proximity Ligation Assay (PLA):

  • Visualize protein interactions in situ at single-molecule resolution

  • Particularly useful for detecting CARD18-caspase-1 interactions in tissue samples

  • Provides spatial information about where interactions occur within cells

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag CARD18 and caspase-1 with appropriate fluorophores

  • Measure energy transfer to detect close proximity

  • Can be used in live cells to monitor dynamic interactions

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein complementation assay

  • Fusion proteins comprising CARD18 and potential partners

  • Fluorescence occurs only when proteins interact

• Yeast Two-Hybrid Screening:

  • Identify novel interaction partners of CARD18

  • Validate interactions using more direct methods

• Surface Plasmon Resonance (SPR):

  • Determine binding kinetics and affinity constants

  • Requires purified recombinant proteins

  • Provides quantitative information about interaction strength

Research has established that CARD18 inhibits generation of IL-1β by interacting with caspase-1 and preventing its association with RIP2 . When designing interaction studies, it's important to consider that the CARD domain is crucial for these interactions, and mutations in this domain may disrupt binding capacity.

What are effective approaches for CARD18 knockdown or knockout to study its function?

To investigate the functional role of CARD18, researchers can employ several approaches to reduce or eliminate its expression:

• siRNA/shRNA-mediated knockdown:

  • Transient siRNA transfection in keratinocyte cell models

  • Stable shRNA expression for long-term studies

  • Previous studies have successfully suppressed CARD18 in skin equivalent cultures using siRNAs without impairing stratum corneum formation

• CRISPR/Cas9 genome editing:

  • Generate complete CARD18 knockout cell lines

  • Create cells with specific mutations in the CARD domain

  • Can be applied to keratinocyte lines or primary cells

• Dominant-negative approaches:

  • Overexpress mutated versions of CARD18 that bind partners but don't function

  • Useful for studying mechanisms of CARD18 action

• Inducible expression systems:

  • Tet-on/off systems to control CARD18 expression

  • Allows temporal control to study acute vs. chronic effects

When designing functional studies, researchers should include appropriate readouts to assess the impact of CARD18 modulation:

• IL-1β production and secretion

• Caspase-1 activation

• Inflammasome assembly

• Keratinocyte differentiation markers

• Response to UV irradiation

• Inflammatory gene expression profiles

Validation of knockdown efficiency should be performed at both mRNA level (qRT-PCR) and protein level (Western blot) using validated CARD18 antibodies .