CAR8 Antibody

What is the CAR8/CA8 antibody and what protein does it target?

Anti-Car8 antibodies (also known as CA8 antibodies) are research tools designed to detect the protein encoded by the gene CA8, or 'carbonic anhydrase 8' in humans. The target protein is a 290-amino acid protein that belongs to the Alpha-carbonic anhydrase family . Despite its classification, CARP VIII (Carbonic Anhydrase-Related Protein VIII) differs from typical carbonic anhydrases as it lacks the classical carbonic anhydrase activity due to the absence of zinc-binding histidine residues . These antibodies serve as crucial research tools for investigating CARP VIII expression and function in both normal physiology and pathological conditions.

Where is CARP VIII/CA8 protein predominantly expressed in normal tissues?

Human CARP VIII is preferentially expressed in the brain, with particularly high expression in Purkinje cells of the cerebellum . This highly specific expression pattern makes it an excellent marker for studying cerebellar function and pathology. Beyond Purkinje cells, little or no expression of CARP VIII has been described in adult human tissue , highlighting its relatively restricted expression pattern. This selective distribution suggests specialized functions for CARP VIII in cerebellar physiology.

What are the common applications for CAR8/CA8 antibodies in research?

Based on available data, common research applications for CAR8/CA8 antibodies include:

What is the molecular weight of the CARP VIII/CA8 protein?

According to immunoblot data, CARP VIII has a molecular weight of approximately 29 kDa . This molecular weight is consistent with the expected size based on its 290-amino acid sequence . When working with CAR8/CA8 antibodies in applications like Western blotting, researchers should expect to detect a band at approximately this size, which serves as one confirmation of antibody specificity.

What species reactivity should be considered when selecting CAR8/CA8 antibodies?

When selecting CAR8/CA8 antibodies for research, it's important to consider their validated reactivity across species. Common reactivity profiles include:

• Human (Hu) - Most widely validated

• Mouse (Ms) - Common for neuroscience research

• Rat (Rt) - Frequently used in neurological models

• Arabidopsis (Ar) - Some antibodies show cross-reactivity
  Species reactivity information is typically provided by antibody suppliers and should be verified experimentally when studying a new species model.

What is the association between CARP VIII antibodies and paraneoplastic cerebellar degeneration?

CARP VIII antibodies have been identified in patients with paraneoplastic cerebellar degeneration (PCD), a condition characterized by subacute cerebellar ataxia caused by widespread loss of Purkinje cells in the cerebellum . In documented case studies, CARP VIII antibodies were detected in:

• A patient with PCD associated with melanoma

• A patient with PCD associated with ovarian adenocarcinoma
  These antibodies target intracellular Purkinje cell proteins, potentially triggering an antigen-specific cytotoxic T-cell response that contributes to Purkinje cell death . In one documented case, despite treatment with chemotherapy and intravenous immunoglobulin, the cerebellar syndrome worsened over six months, and brain MRI showed progressive cerebellar atrophy .

How is CARP VIII expression altered in cancer cells compared to normal tissues?

While CARP VIII expression is highly restricted in normal adult tissues (primarily to cerebellar Purkinje cells), it becomes strongly upregulated in various cancer types . This expression pattern alteration makes it potentially valuable as a tumor marker.
Documented cancer types with CARP VIII upregulation include:

• Ovarian carcinoma

• Colorectal carcinoma

• Non-small-cell lung carcinoma

• Melanoma (implied from association with PCD)
  Immunohistochemical analysis of tumor biopsy material using commercial anti-CARP VIII antibodies has demonstrated robust expression of CARP VIII in tumor cells . This aberrant expression may explain the development of autoantibodies in some cancer patients, particularly those who develop paraneoplastic neurological syndromes.

How can epitope mapping be performed for CARP VIII antibodies?

Epitope mapping for CARP VIII antibodies can be accomplished through several sophisticated approaches:

• Phage Display Technology: Express fragments of CARP VIII on phage surfaces and screen with test antibodies to identify binding regions. This approach has been successfully used to map epitopes recognized by autoantibodies in patients with PCD .

• Competitive Inhibition Assays: Pre-incubate tissue sections with known CARP VIII antibodies before adding test antibodies. Competition for binding indicates shared or nearby epitopes. For example, biotinylated anti-CARP VIII IgG failed to immunoreact with brain sections pre-incubated with patient serum containing CARP VIII antibodies, indicating competition for the same epitopes .

• Deletion Mutants Analysis: Create a series of CARP VIII deletion constructs to isolate specific binding regions, allowing precise mapping of antibody recognition sites.

• Peptide Scanning: Synthesize overlapping peptides spanning the CARP VIII sequence and test for antibody binding to identify specific recognition sequences.
  These approaches are particularly valuable for characterizing autoantibodies in neurological disorders and for validating research antibodies.

What methodological approaches exist for studying CARP VIII in neurodegenerative disorders?

For investigating CARP VIII in neurodegenerative contexts, several specialized methodological approaches have proven effective:

• Cerebellar Slice Immunohistochemistry: Provides high-resolution visualization of CARP VIII distribution within the complex cerebellar architecture and allows quantification of Purkinje cell loss .

• Dual Immunofluorescence: Combining CARP VIII antibodies with markers of neurodegeneration (caspase-3, TUNEL) or inflammation (microglial markers) can reveal pathological mechanisms affecting Purkinje cells.

• CSF Antibody Profiling: Testing for anti-CARP VIII antibodies in cerebrospinal fluid can help identify autoimmune contributions to cerebellar degeneration .

• Patient-Derived Autoantibody Studies: Purifying IgG from patients with cerebellar ataxia for use in experimental models can help establish pathogenicity mechanisms .

• Competitive Inhibition Assays: These can determine if patient antibodies recognize the same epitopes as known CARP VIII antibodies, providing insight into disease-specific immune responses .
  These approaches have yielded valuable information about conditions like paraneoplastic cerebellar degeneration where CARP VIII autoimmunity plays a role.

How do CARP VIII antibodies compare with other onconeural antibodies in neurological disorders?

CARP VIII antibodies represent a distinct category among onconeural antibodies associated with neurological presentations:

What are the optimal fixation and immunohistochemistry protocols for CAR8/CA8 antibodies?

For optimal immunohistochemical detection of CARP VIII, consider the following protocol recommendations:

• Tissue Preparation:

  • For paraffin sections: Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • For frozen sections: Fix in 4% paraformaldehyde for 15-20 minutes

• Antigen Retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-100°C for 20 minutes

  • Allow gradual cooling to room temperature

• Blocking and Antibody Application:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Incubate with primary anti-CARP VIII antibody at optimized dilution (typically 1:100-1:500)

  • Overnight incubation at 4°C generally yields superior results

• Detection Systems:

  • For chromogenic detection: HRP-conjugated secondary antibody with DAB substrate

  • For fluorescent detection: Fluorophore-conjugated secondary antibodies

• Counterstaining:

  • Light hematoxylin counterstain for chromogenic detection

  • DAPI nuclear counterstain for fluorescent detection
    These protocols should be optimized for each specific anti-CARP VIII antibody, as different clones may have different optimal conditions .

How can specificity of CAR8/CA8 antibodies be validated in experimental procedures?

Rigorous validation of CAR8/CA8 antibody specificity is essential for reliable research outcomes. Recommended validation approaches include:

• Western Blot Analysis: Confirm detection of a single band at the expected molecular weight of approximately 29 kDa in cerebellar lysates .

• Phage Plaque Screening: Test antibodies against phage plaques expressing CARP VIII and control plaques (which do not express CARP VIII). Specific antibodies should only stain CARP VIII-expressing plaques .

• Competitive Inhibition Assays: Pre-incubate tissue sections with serum containing confirmed anti-CARP VIII antibodies before applying test antibodies. Loss of staining indicates competition for the same epitopes .

• Immunohistochemical Pattern Recognition: Verify the expected staining pattern of selective Purkinje cell labeling in cerebellar tissue .

• Multiple Antibody Concordance: Compare staining patterns using different antibodies targeting distinct CARP VIII epitopes.

• Absorbing Antibodies with Purified Antigen: Pre-incubate antibodies with purified CARP VIII protein to confirm specific binding.
  These validation steps ensure that experimental findings accurately reflect CARP VIII biology rather than non-specific interactions.

What are the key considerations for selecting monoclonal versus polyclonal CAR8/CA8 antibodies?

The choice between monoclonal and polyclonal antibodies for CARP VIII research should be guided by specific experimental requirements:

• For quantitative Western blotting: Monoclonals provide better standardization

• For detecting CARP VIII in fixed tissues: Polyclonals may offer superior sensitivity

• For co-localization studies: Monoclonals from different species facilitate dual labeling

• For detecting denatured proteins: Polyclonals generally perform better
  The final selection should be based on the specific research question, required applications, and importance of standardization versus sensitivity .

What controls should be included when using CAR8/CA8 antibodies in Western blotting?

Robust controls are essential for reliable Western blotting with CAR8/CA8 antibodies:

• Positive Tissue Control: Cerebellar tissue lysate should show a strong band at approximately 29 kDa, reflecting the high expression of CARP VIII in Purkinje cells .

• Negative Tissue Control: Tissues known to lack CARP VIII expression should be included to confirm specificity.

• Molecular Weight Verification: Use high-quality molecular weight markers to confirm the detected band appears at the expected size of approximately 29 kDa .

• Loading Control: Include antibodies against housekeeping proteins (β-actin, GAPDH) to normalize for protein loading variations.

• Peptide Competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific signal.

• Secondary Antibody Control: Omit primary antibody to identify non-specific secondary antibody binding.

• Sample Preparation Control: Include both reducing and non-reducing conditions if studying potential multimeric forms.
  These controls help distinguish specific signal from artifacts and ensure proper interpretation of results, particularly important when studying proteins with restricted expression patterns like CARP VIII .

How can immunofluorescence techniques be optimized for dual labeling with CAR8/CA8 antibodies?

For optimal dual immunofluorescence labeling involving CARP VIII antibodies, consider these methodological refinements:

• Primary Antibody Selection:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-CARP VIII with mouse anti-calbindin)

  • If using same-species antibodies, consider directly conjugated antibodies or sequential labeling protocols

• Signal Optimization:

  • Begin with the less abundant target or weaker antibody first

  • For CARP VIII, use tyramide signal amplification if detecting low expression levels outside cerebellum

• Cross-Reactivity Prevention:

  • Block with sera from both secondary antibody host species

  • Include additional blocking step after first secondary antibody application

  • Use highly cross-adsorbed secondary antibodies

• Sequential Protocol Example:

  • Apply anti-CARP VIII antibody and detect with species-specific secondary antibody

  • Block unoccupied binding sites with excess irrelevant antibody from same species

  • Apply second primary antibody and detect with differently labeled secondary antibody

• Controls:

  • Single-antibody controls to establish baseline signals

  • Secondary-only controls to assess background

  • Reversed sequence controls to verify no cross-detection
    These protocols facilitate co-localization studies of CARP VIII with neuronal markers, synaptic proteins, or pathological markers in both normal and disease contexts .