CARD6 Antibody

What is CARD6 and what cellular functions is it involved in?

CARD6 is a 1,037-amino acid (human) or 1,175-amino acid (mouse) protein containing a caspase recruitment domain (CARD) at the N-terminus, a glutamic acid-rich region following the CARD, and a proline-rich region at the C-terminus . Structurally, CARD6 shares similarities with interferon (IFN)-inducible GTPases .

Functionally, CARD6:

• Associates with microtubules in a CARD- and proline-rich region-dependent manner

• Interacts with receptor-interacting protein 2 (RIP2), a mediator of NF-κB activation induced by NOD receptors

• Shows enhanced expression in response to beta IFN and gamma IFN

• May participate in NF-κB signaling pathways important for immune responses

Research has demonstrated that CARD6 expression is upregulated in several cancer types, including esophageal squamous cell carcinoma (70.7% of cases), gastric adenocarcinomas (45.0%), and colorectal adenocarcinomas (78.6%), compared to much lower expression in corresponding normal tissues .

What types of CARD6 antibodies are commonly available for research?

Several types of CARD6 antibodies are available for research applications:

Most commercially available CARD6 antibodies are rabbit polyclonal antibodies targeting human CARD6, though some cross-react with mouse and rat CARD6 .

What are the standard applications for CARD6 antibodies?

CARD6 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Detects CARD6 protein (predicted band size: ~116 kDa) in cell and tissue lysates

• Immunohistochemistry (IHC): Visualizes CARD6 expression in paraffin-embedded tissues, useful for cancer studies

• Immunocytochemistry/Immunofluorescence (ICC/IF): Localizes CARD6 within cells, showing its association with microtubules

• Cell-based ELISA: Quantitatively determines CARD6 expression levels in cultured cells

The subcellular localization pattern of CARD6 varies by cell type, with immunostaining observed in the nucleus/cytoplasm of esophageal squamous cell carcinoma or predominantly in the cytoplasm of gastric and colorectal cancer cells .

How should researchers validate CARD6 antibodies for their specific experimental systems?

Proper validation of CARD6 antibodies is critical for experimental reliability:

• Specificity testing:

  • Western blot analysis should show a single protein band at the expected molecular weight (~116 kDa)

  • Use positive controls (tissues/cells known to express CARD6, such as HeLa cells)

  • Include negative controls (CARD6-deficient cells or tissues)

  • Peptide blocking experiments can confirm specificity, where the immunogen peptide blocks antibody binding

• Cross-reactivity assessment:

  • Test antibodies on lysates from different species if cross-species reactivity is claimed

  • Verify reported species reactivity (many CARD6 antibodies are human-specific, while others cross-react with mouse and rat)

• Application-specific validation:

  • For IHC: Compare staining patterns with published literature

  • For IF: Confirm subcellular localization matches known patterns (microtubule association)

  • For knockdown studies: Confirm reduced signal in CARD6-silenced cells (siRNA)

As emphasized in recent literature on antibody reliability, researchers should be aware that many antibodies used in research do not recognize their intended target or recognize additional molecules, compromising research integrity .

What controls are essential when using CARD6 antibodies in experimental procedures?

For cell-based ELISA assays specifically, the CytoGlow™ CARD6 Colorimetric Cell-Based ELISA Kit recommends:

• Using anti-GAPDH antibody as an internal positive control

• Including HRP-conjugated secondary antibody alone as a negative control (without primary antibody)

• Performing experiments in duplicate or triplicate to ensure reproducibility

How can CARD6 antibodies be used to study protein-protein interactions between CARD6 and its binding partners?

CARD6 interacts with several proteins, most notably RIP2 (receptor-interacting protein 2). Researchers can investigate these interactions using:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate CARD6 using anti-CARD6 antibodies and probe for associated proteins like RIP2

  • Research indicates that the CARD of CARD6, interestingly, negatively controls the coimmunoprecipitation of CARD6 and RIP2

  • Consider using ΔCARD-CARD6 mutants compared to wild-type for differential binding studies

• Proximity ligation assays (PLA):

  • Visualize in situ protein-protein interactions between CARD6 and binding partners

  • Requires primary antibodies from different host species for CARD6 and interacting partner

• Immunofluorescence co-localization:

  • CARD6 associates with microtubules in a CARD- and proline-rich region-dependent manner

  • Double immunofluorescence staining with CARD6 antibody and microtubule markers can visualize this association

Research has shown that CARD6's interaction with RIP2 is complex: "CARD6 associates with microtubules and with receptor-interacting protein 2 (RIP2). RIP2 mediates NF-κB activation induced by the intracellular nucleotide-binding oligomerization domain (NOD) receptors that sense bacterial peptidoglycan."

How does CARD6 expression and localization change in response to immune stimulation?

CARD6 expression is regulated by immune stimuli, making antibody-based detection valuable for studying immune responses:

• Interferon regulation:

  • CARD6 expression in bone marrow-derived macrophages is rapidly induced by beta IFN and gamma IFN

  • IFN-induced upregulation of CARD6 is suppressed by lipopolysaccharide (LPS), in contrast to LPS's enhancement of IFN-induced RIP2 upregulation

  • Researchers can use CARD6 antibodies in time-course experiments to track expression changes after IFN treatment

• Subcellular trafficking:

  • The CARD of RIP2 mediates the translocation of CARD6 to aggresomes

  • Immunofluorescence experiments can track this redistribution using CARD6 antibodies

• Stress responses:

  • In spinal cord injury models, CARD6 levels change, and CARD6 knockdown affects neuroinflammatory responses

  • IHC with CARD6 antibodies can characterize these changes in tissue sections

What technical challenges might researchers encounter when using CARD6 antibodies, and how can these be addressed?

Recent literature highlights that "Antibodies are one of the most important reagents used in biomedical and fundamental research... Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings." This emphasizes the importance of proper validation and controls when using CARD6 antibodies.

How are CARD6 antibodies being utilized in cancer research?

CARD6 has emerged as a protein of interest in oncology research:

• Expression profiling in cancer:

  • Studies using CARD6 antibodies have demonstrated that CARD6 is expressed in cancer cells of esophageal squamous cell carcinoma (ESCC) (70.7%), gastric adenocarcinomas (GC) (45.0%), and colorectal adenocarcinomas (CRC) (78.6%)

  • Notably, in gastric cancer, intestinal-type GC (77.8%) showed higher expression of CARD6 than diffuse-type GC (20.0%) and mixed-type GC (50.0%)

  • Normal epithelial cells show much lower frequencies of CARD6 immunostaining: oesophagus (0%), stomach (8.0%), and colon (5.0%)

• NF-κB signaling in cancer:

  • "The increased expression of CARD6 in ESCC, GC and CRC tissues compared to their corresponding normal cells suggested that neoexpression of CARD6 might be related to activation of NF-κB pathway in the cancers and might play a role in the development of most types of gastrointestinal cancers."

  • Researchers can use CARD6 antibodies to study its relationship with NF-κB pathway components in cancer cells

• Prognostic biomarker potential:

  • CARD6 expression is evident from early TNM stages (stage I) , suggesting potential utility as an early diagnostic marker

  • IHC with CARD6 antibodies can be used to evaluate CARD6 as a potential biomarker

What role does CARD6 play in neuroinflammation and how can antibodies help study this function?

Research has implicated CARD6 in neuroinflammatory processes:

• Spinal cord injury studies:

  • "CARD6 was identified as a suppressor of SCI in mice. CARD6 knockout significantly accelerated functional deficits, neuron death and glia activation."

  • CARD6 knockdown in BV2 cells showed stronger intensity and expression of Iba-1 after LPS stimulation, indicating greater microglial activation

• Apoptotic signaling:

  • "Stronger immunoreactivity of cytosolic Cyto-c and higher levels of TUNEL-positive cells were observed in CARD6 knockdown BV2 cells following LPS exposure"

  • "LPS-induced release of Cyto-c from mitochondria to cytoplasm was markedly accelerated in CARD6 knockdown cells, accompanied with higher expression of Bax and cleaved Caspase-3"

• Research applications:

  • Anti-CARD6 antibodies can be used in co-localization studies with markers of neuroinflammation

  • Western blotting with CARD6 antibodies can track protein level changes in injury models

  • Immunoprecipitation with CARD6 antibodies can help identify binding partners in neural cells

What are the optimal protocols for using CARD6 antibodies in Western blot applications?

Optimized Western Blot Protocol for CARD6 Detection:

• Sample preparation:

  • Extract proteins from cells/tissues using RIPA buffer with protease inhibitors

  • Use fresh samples when possible to minimize degradation

  • Determine protein concentration (BCA or Bradford assay)

  • Load 30 μg of protein per lane (based on successful detection in A549 cell lysate)

• Gel electrophoresis:

  • Use 7.5% SDS-PAGE for better resolution of high molecular weight CARD6 (~116 kDa)

  • Include positive control (A549 or HeLa cell lysate)

• Transfer and blocking:

  • Transfer proteins to PVDF membrane (recommended over nitrocellulose for high MW proteins)

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

• Primary antibody incubation:

  • Dilute anti-CARD6 antibody 1:5000 in primary antibody diluent

  • Incubate overnight at 4°C with gentle rocking

• Secondary antibody and detection:

  • Wash membrane 3x with TBST

  • Incubate with HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000)

  • Develop using ECL substrate

  • Expected band size: 116 kDa

How can researchers optimize immunohistochemistry protocols for CARD6 detection in tissue samples?

Optimized IHC Protocol for CARD6 Detection in Paraffin-Embedded Tissues:

• Tissue preparation:

  • Section formalin-fixed paraffin-embedded tissues at 4-5 μm thickness

  • Mount on positively charged slides

• Antigen retrieval:

  • Deparaffinize and rehydrate sections through xylene and graded alcohols

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with 5% normal goat serum

  • Incubate with anti-CARD6 antibody at 1:500 dilution overnight at 4°C

• Detection system:

  • Apply HRP-conjugated secondary antibody

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount

• Controls and interpretation:

  • Include positive control tissue (gastric cancer tissue has shown reliable staining)

  • Include negative control (omit primary antibody)

  • CARD6 staining may appear in nucleus/cytoplasm (ESCC) or predominantly cytoplasm (GC and CRC)

What cell-based assay methods can be used with CARD6 antibodies to study its function?

Several cell-based assay approaches can be used with CARD6 antibodies:

• Colorimetric Cell-Based ELISA:

  • The CytoGlow™ CARD6 Colorimetric Cell-Based ELISA allows for detection of CARD6 and monitoring expression changes under different treatments

  • This assay uses indirect ELISA format where target protein is captured by target-specific primary antibodies and detected with HRP-conjugated secondary antibodies

  • Protocol includes cell fixation, quenching, blocking, primary and secondary antibody incubation, substrate development, and optional Crystal Violet cell staining

• siRNA-mediated knockdown validation:

  • Transfect cells with CARD6-specific siRNA and control siRNA

  • Confirm knockdown efficiency using CARD6 antibodies in Western blot

  • Assess functional consequences using appropriate assays

• Immunofluorescence for localization studies:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 1% BSA

  • Incubate with anti-CARD6 antibody (1:500 dilution)

  • Visualize using fluorophore-conjugated secondary antibodies

  • Counterstain nucleus with Hoechst 33342