CARD11 Antibody

What is CARD11 and what is its role in immune signaling?

CARD11, also known as CARMA1 or BIMP3, is a 1154 amino acid scaffold protein that plays a critical role in adaptive immune response by transducing the activation of NF-κB downstream of T-cell receptor (TCR) and B-cell receptor (BCR) engagement . CARD11 contains multiple functional domains including a caspase recruitment domain (CARD, amino acids 1-110), LATCH (112-130), coiled-coil (CC, 130-449) domains, and a C-terminal membrane-associated guanylate kinase domain (MAGUK, 667-1140) comprised of PDZ, SH3, and GUK domains .

Upon TCR or BCR activation, CARD11 homooligomerizes to form a nucleating helical template that recruits BCL10 via CARD-CARD interaction, promoting polymerization of BCL10 and subsequent recruitment of MALT1. This leads to I-kappa-B kinase (IKK) phosphorylation and degradation, releasing NF-κB proteins for nuclear translocation . CARD11 also activates the TORC1 signaling pathway and promotes linear ubiquitination of BCL10 by targeting it to RNF31/HOIP .

What types of CARD11 antibodies are available for research applications?

CARD11 antibodies are available in different formats, with polyclonal antibodies being common. For example, the 21741-1-AP is a rabbit polyclonal antibody that targets CARD11 in various applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and ELISA . Similarly, ab264296 is a rabbit polyclonal antibody suitable for immunoprecipitation (IP) and Western Blot applications .

These antibodies are typically generated using specific immunogens, such as CARD11 fusion proteins or synthetic peptides corresponding to regions within the human CARD11 protein. For instance, ab264296 uses an immunogen corresponding to a synthetic peptide within human CARD11 amino acids 450-550 .

What are the recommended storage conditions for CARD11 antibodies?

For optimal performance and longevity, CARD11 antibodies should be stored at -20°C. Under these conditions, most antibodies remain stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

What are the recommended applications and dilutions for CARD11 antibody use?

CARD11 antibodies can be used across multiple application platforms with specific recommended dilutions:

It is important to note that these dilutions serve as starting points, and researchers should titrate the antibody in each testing system to obtain optimal results as the effectiveness may be sample-dependent . Published applications have demonstrated successful use of certain CARD11 antibodies in Western Blot and Immunofluorescence applications across multiple peer-reviewed studies .

What cell and tissue types are appropriate for CARD11 detection?

CARD11 antibodies have demonstrated positive detection in various cell and tissue types:

For immunohistochemistry applications, antigen retrieval is an important consideration. The recommended protocol suggests using TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative .

CARD11 expression is primarily detected in adult peripheral blood leukocytes, thymus, and spleen, making these tissues particularly relevant for research involving immune system functions .

How can I validate the specificity of my CARD11 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For CARD11 antibodies, multiple validation approaches should be employed:

• Western blot analysis with positive controls: Use Raji cells or mouse thymus tissue lysates, where CARD11 should be detected at approximately 133 kDa (matching the observed molecular weight) .

• Knockout/knockdown validation: Compare detection in wild-type samples versus those where CARD11 has been silenced using siRNA, as demonstrated in studies of collagen-induced arthritis where CARD11 siRNA effectively reduced CARD11 expression .

• Immunoprecipitation analysis: Verify the antibody's ability to precipitate CARD11 and its interaction partners like BCL10, which can confirm both specificity and functionality in detecting protein complexes .

• Cross-reactivity testing: Confirm species reactivity matches the manufacturer's claims. For example, the 21741-1-AP antibody shows reactivity with human, mouse, and rat samples .

How is CARD11 implicated in rheumatoid arthritis research?

CARD11 plays a significant role in the pathogenesis of rheumatoid arthritis (RA) through its involvement in inflammatory signaling pathways. Research using collagen-induced arthritis (CIA) mouse models has demonstrated that systemic administration of CARD11 siRNA significantly reduces clinical scores of arthritis severity .

Histological analyses show that CARD11 silencing attenuates joint inflammation and destruction. This is further supported by microcomputed tomography (micro-CT) findings showing less severe joint destruction in CARD11 siRNA-treated mice compared to controls .

The mechanism appears to involve inhibition of several key inflammatory processes:

• Reduced formation of the CARD11/Bcl10 complex

• Decreased nuclear factor-kappa B (NF-κB) activation

• Significant reduction in proinflammatory cytokines IL-1β, IL-6, and IL-17

• Lowered serum anti-type II collagen (anti-CII) antibody levels

• Decreased percentage of T helper type 17 (Th17) cells

These findings suggest CARD11 as a potential therapeutic target for rheumatoid arthritis treatment strategies.

What is known about CARD11 mutations in primary immune disorders?

CARD11 mutations are associated with several distinct primary immune disorders, with the functional impact of the mutation determining the specific clinical presentation:

• Biallelic null mutations: Cause severe combined immunodeficiency (SCID) .

• Heterozygous gain-of-function mutations: Lead to B cell Expansion with NF-κB and T cell Anergy (BENTA), characterized by polyclonal expansion of B cells, lymphadenopathy, and splenomegaly .

• Heterozygous loss-of-function, dominant interfering mutations: Associated with severe atopic disease .

• Novel heterozygous mutations: Recently identified mutations extend beyond atopy to include diverse immunologic phenotypes resembling STAT3-LOF, DOCK8 deficiency, common variable immune deficiency (CVID), neutropenia, and IPEX-like syndrome .

A recent case report identified a novel germline in-frame three base-pair deletion (c.1030_1032del, p.K344del) in the CARD11 gene in a Chinese patient with atypical BENTA, presenting with recurrent fever and B cell lymphocytosis . To date, 23 patients with BENTA have been identified carrying seven distinct gain-of-function mutations: C49Y, G123S, G123D, G126D, E134G, H234Ldel235-8, and K215del .

How can CARD11 antibodies be used to study CARD11/Bcl10 complex formation?

The CARD11/Bcl10 complex formation is a critical step in NF-κB signaling pathway activation. CARD11 antibodies can be effectively employed to study this complex using the following methodological approaches:

• Co-immunoprecipitation (Co-IP): CARD11 antibodies can be used to immunoprecipitate CARD11 and its binding partners, allowing researchers to assess the formation of the CARD11/Bcl10 complex under various experimental conditions. This technique was successfully employed in a study demonstrating that CARD11 siRNA treatment inhibited the formation of the CARD11/Bcl10 complex in splenocytes from CIA mice .

• Immunoblotting following Co-IP: After immunoprecipitation with CARD11 antibodies, researchers can use immunoblotting with Bcl10 antibodies to detect and quantify the amount of Bcl10 associated with CARD11, providing a measure of complex formation .

• Microscopy techniques: Combining CARD11 antibodies with Bcl10 antibodies in immunofluorescence studies can visualize the co-localization of these proteins, particularly following T-cell or B-cell receptor stimulation.

• Proximity ligation assays: This method can detect protein-protein interactions using two antibodies (CARD11 and Bcl10) and generate fluorescent signals only when the proteins are in close proximity, allowing for precise spatial assessment of complex formation in intact cells.

How do CARD11 mutations affect NF-κB signaling pathways in lymphocytes?

CARD11 mutations can have diverse effects on NF-κB signaling depending on their functional consequences. Research has revealed three primary categories of mutations with distinct molecular mechanisms:

• Gain-of-function mutations: These mutations (such as those found in BENTA syndrome) lead to constitutive NF-κB activation independent of antigen receptor stimulation. This results in excessive B cell proliferation but paradoxically causes T cell anergy. The molecular mechanism involves enhanced CARD11 oligomerization and increased recruitment of BCL10, leading to persistent downstream signaling .

• Loss-of-function mutations: Complete loss of CARD11 function prevents NF-κB activation following antigen receptor engagement, resulting in severe combined immunodeficiency due to impaired lymphocyte development and function .

• Dominant-negative mutations: These mutations (often found in patients with severe atopy) allow CARD11 protein production but interfere with normal signaling. Mechanistically, these mutant proteins can still interact with wildtype CARD11 and other signaling components but fail to propagate the signal, effectively inhibiting NF-κB activation in a dominant-negative manner .

Experimental approaches to study these effects include reconstituting CARD11-deficient cell lines with mutant CARD11 constructs and measuring NF-κB activation through reporter assays, phosphorylation status of signaling components, and nuclear translocation of NF-κB subunits.

What experimental approaches can quantify CARD11's impact on inflammatory cytokine production?

Several experimental approaches can effectively quantify CARD11's impact on inflammatory cytokine production:

• ELISA assays: Enzyme-linked immunosorbent assays can measure cytokine levels in serum and tissue homogenates. Research has demonstrated that CARD11 silencing significantly reduces levels of IL-1β, IL-6, and IL-17 in both serum and homogenized joints of CIA mice .

• Flow cytometry: This technique can assess intracellular cytokine production at the single-cell level. For example, flow cytometry has been used to demonstrate that CARD11 siRNA treatment reduces the percentage of Th17 cells (CD4+IL-17+) in splenocytes compared to controls .

• qRT-PCR: Quantitative reverse transcription PCR can measure cytokine gene expression at the mRNA level, providing insight into the transcriptional effects of CARD11 manipulation.

• Multiplex cytokine assays: These assays allow simultaneous quantification of multiple cytokines from a single sample, providing a more comprehensive profile of the inflammatory environment.

• In vitro stimulation assays: Isolated lymphocytes can be stimulated with specific antigens or mitogens in the presence or absence of CARD11 inhibition, and subsequent cytokine production can be measured to directly assess CARD11's role in the response.

What considerations should be made when using CARD11 antibodies in co-localization studies?

When conducting co-localization studies using CARD11 antibodies, several important methodological considerations should be addressed:

• Antibody compatibility: When performing dual or multi-color staining, ensure that the CARD11 antibody is compatible with other antibodies in terms of species origin and detection systems. For example, if using rabbit polyclonal CARD11 antibodies, other primary antibodies should be from different species to avoid cross-reactivity .

• Subcellular localization: CARD11 primarily localizes in the cytoplasm and can co-localize with DPP4 in membrane rafts . When designing experiments, consider appropriate fixation and permeabilization protocols that preserve these structures while allowing antibody access.

• Stimulation conditions: CARD11's localization and interaction patterns change upon T-cell or B-cell receptor stimulation. Time-course experiments following activation may be necessary to capture dynamic changes in protein interactions.

• Resolution limitations: Standard fluorescence microscopy may not provide sufficient resolution to distinguish between true co-localization and coincidental proximity. Consider super-resolution microscopy techniques or proximity ligation assays for more definitive results.

• Positive controls: Include known CARD11 interaction partners (such as BCL10) as positive controls for co-localization studies .

• Quantification methods: Implement objective quantification methods for co-localization using appropriate software and statistical analyses rather than relying solely on visual assessment.

What are common challenges in detecting CARD11 in tissue samples?

Detecting CARD11 in tissue samples can present several challenges that researchers should address methodically:

• Antigen retrieval optimization: CARD11 detection in tissues often requires specific antigen retrieval conditions. For optimal results with IHC applications, TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 can serve as an alternative . Insufficient or inappropriate antigen retrieval can lead to false negative results.

• Tissue-specific expression levels: CARD11 is primarily expressed in lymphoid tissues such as peripheral blood leukocytes, thymus, and spleen . Detection in other tissues may require more sensitive methods or higher antibody concentrations.

• Specificity in lymphoma tissues: While CARD11 antibodies have shown positive IHC detection in human lymphoma tissues , the heterogeneity of lymphomas can affect detection consistency. Researchers should validate antibody performance in their specific lymphoma subtype of interest.

• Fixation effects: Overfixation can mask epitopes and reduce antibody binding. Optimization of fixation protocols (duration, fixative type) may be necessary for consistent CARD11 detection.

• Background reduction: Nonspecific binding can obscure specific CARD11 signals. Implement appropriate blocking steps (using BSA or serum) and optimize antibody dilutions to improve signal-to-noise ratio.

How can Western blot protocols be optimized for CARD11 detection?

Optimizing Western blot protocols for CARD11 detection requires attention to several key parameters:

• Sample preparation: CARD11 is a relatively large protein (133 kDa observed molecular weight) , which can be susceptible to degradation. Use fresh samples when possible and include protease inhibitors in lysis buffers to prevent degradation.

• Gel percentage selection: Due to CARD11's high molecular weight, use lower percentage gels (6-8%) to ensure proper resolution and separation from other high molecular weight proteins.

• Transfer conditions: Longer transfer times or specialized protocols for high molecular weight proteins may be necessary. Consider wet transfer methods rather than semi-dry transfer for improved efficiency of larger proteins.

• Antibody dilution optimization: Start with the recommended 1:500-1:1000 dilution range , but perform titration experiments to determine the optimal concentration for your specific sample type and detection system.

• Positive controls: Include known CARD11-expressing samples such as Raji cells or mouse thymus tissue lysates as positive controls to validate detection.

• Incubation conditions: Optimize primary antibody incubation time and temperature. For high molecular weight proteins like CARD11, longer incubation times (overnight at 4°C) often yield better results than shorter incubations at room temperature.

• Detection system sensitivity: Choose a detection system with appropriate sensitivity based on the expected expression level of CARD11 in your samples. Enhanced chemiluminescence (ECL) systems with longer exposure times may be necessary for samples with lower expression.