CABP4 Antibody

What is CABP4 and what is its structural composition?

CABP4 is a neuronal calcium-binding protein belonging to the calmodulin (CaM) superfamily that regulates calcium channel targets in the brain and retina. Structurally, it contains two globular domains (N- and C-lobe), each containing a pair of EF-hand motifs connected by a central linker. The second EF-hand in CABP4 lacks conserved residues in the binding loop and is predicted to not bind Ca²⁺ . The protein is characterized by four EF-hand motifs, though not all may be functional for calcium binding .

The full-length CABP4 protein contains specific regions that are targeted by different antibodies, including regions at the N-terminus (AA 1-107, AA 1-170), internal regions, and C-terminal regions (AA 175-203) . This structural organization is important when selecting antibody epitopes for experimental applications.

What is the primary function of CABP4 in neuronal tissues?

CABP4 plays an essential role in regulating voltage-gated calcium channels, particularly in retinal photoreceptor cells. It localizes at photoreceptor synaptic terminals in both rods and cones, where it modulates the function of Cav1.4 L-type voltage-dependent calcium channels . This calcium-dependent regulation is crucial for:

• Development and maintenance of photoreceptor synapses

• Modulation of neurotransmitter release

• Proper calcium signaling in retinal neurons

CABP4 exerts its regulatory function through calcium-dependent structural interaction with the C-terminal region of Cav1.4, influencing voltage-dependent activation of these channels . This interaction is critical for normal visual processing, as evidenced by the association between CABP4 mutations and visual disorders.

What disease associations have been established for CABP4 mutations?

CABP4 mutations have been linked to multiple neurological conditions:

• Congenital Stationary Night Blindness (CSNB2): Multiple studies have established that mutations in both Cav1.4 and CABP4 can cause CSNB2, with mouse models lacking CABP4 displaying CSNB2-like phenotypes .

• Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE): More recent research has identified a novel missense mutation in CABP4 (c.464G>A, p.G155D) in a 4-generation pedigree with ADNFLE, establishing a potential new pathogenic mechanism for this form of epilepsy .

The involvement of CABP4 in both visual and broader neurological disorders highlights its importance in calcium signaling throughout the nervous system. Research suggests the G155D mutation reduces CABP4 protein stability while potentially increasing mRNA expression, indicating complex regulatory effects that ultimately influence neuronal excitability .

What are the critical factors to consider when selecting a CABP4 antibody?

When selecting a CABP4 antibody for research applications, consider the following factors:

• Target epitope location: Different antibodies target various regions of CABP4. Available options include antibodies targeting N-terminal regions (AA 1-107, AA 1-170), internal regions, and C-terminal regions (AA 175-203) . The epitope location should be selected based on:

  • Known functional domains

  • Accessibility in your experimental conditions

  • Avoidance of regions affected by mutations in your experimental model

• Species reactivity: Ensure the antibody recognizes CABP4 in your species of interest. Available antibodies demonstrate reactivity to human, mouse, rat, monkey, horse, and dog CABP4, though reactivity profiles vary by product .

• Clonality: Both monoclonal (e.g., 5G11, 3B3) and polyclonal CABP4 antibodies are available . Monoclonal antibodies offer higher specificity for a single epitope, while polyclonal antibodies provide broader detection but potential cross-reactivity.

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, immunohistochemistry, immunofluorescence, etc.) .

How should researchers validate CABP4 antibody specificity?

Proper validation of CABP4 antibody specificity is essential for experimental reliability:

• Blocking peptide experiments: Use specific blocking peptides to confirm antibody specificity. As demonstrated in Western blot analyses, preincubating anti-CABP4 antibody with a specific blocking peptide (e.g., CaBP4 Blocking Peptide #BLP-CS004) should eliminate the specific band observed in target tissues like rat eye lysate .

• Knockout/knockdown controls: When possible, utilize CABP4 knockout or knockdown samples as negative controls to verify antibody specificity.

• Recombinant protein controls: Test antibodies against recombinant CABP4 protein to confirm target recognition in a controlled system.

• Cross-reactivity assessment: Examine potential cross-reactivity with other calcium-binding proteins, particularly those within the CaBP family that share structural similarities with CABP4.

• Tissue-specific expression correlation: Verify that antibody staining patterns correlate with known CABP4 expression patterns, particularly in retinal photoreceptor synaptic terminals .

What are the optimal conditions for Western blot analysis using CABP4 antibodies?

For optimal Western blot results with CABP4 antibodies:

• Sample preparation:

  • Use appropriate tissue sources where CABP4 is expressed (retinal tissue is ideal)

  • Include protease inhibitors in lysis buffers to prevent degradation

  • For mutation studies, consider transfection of human neuronal cells with recombinant CABP4 plasmids (wild-type or mutant)

• Dilution factors:

  • Recommended dilutions vary by product; for example, Anti-CaBP4 Antibody (#ACS-004) has been tested at 1:200 dilution for Western blot of rat eye lysate

  • Optimize antibody concentration through titration experiments

• Detection systems:

  • Secondary antibody selection should match the host species of your primary antibody (commonly rabbit for polyclonal antibodies)

  • Consider enhanced chemiluminescence for sensitive detection of low-abundance CABP4

• Controls:

  • Include peptide-blocked antibody controls to verify specificity

  • When studying mutations, compare wild-type and mutant CABP4 expression

How can CABP4 antibodies be effectively used in studying disease-associated mutations?

CABP4 antibodies can be powerful tools for studying disease-associated mutations through these approaches:

• Expression analysis: Western blot analysis can reveal differences in protein expression levels between wild-type and mutant CABP4. For example, the c.464G>A (p.G155D) mutation associated with ADNFLE shows reduced protein expression despite increased mRNA levels, suggesting decreased protein stability .

• Cellular localization studies: Immunofluorescence using CABP4 antibodies can determine whether mutations affect proper cellular localization, particularly at photoreceptor synaptic terminals.

• Protein-protein interaction assessment: Co-immunoprecipitation using CABP4 antibodies can evaluate how mutations affect interactions with binding partners like Cav1.4 calcium channels.

• In vitro model systems: Transfection of neuronal cells with recombinant plasmids expressing wild-type or mutant CABP4, followed by antibody-based detection, allows detailed comparison of protein function and stability .

• Correlation with electrophysiological data: Combine CABP4 antibody studies with patch-clamp recordings to correlate protein expression with functional changes in calcium channel activity and neuronal excitability .

How do CABP4 antibodies contribute to understanding calcium channel modulation?

CABP4 antibodies enable detailed investigation of calcium channel regulation mechanisms:

• Structural interaction studies: CABP4 has been shown to interact with the C-terminal region of Cav1.4 in a calcium-dependent manner, modulating voltage-dependent channel activation . Antibodies can help identify specific interaction domains through co-immunoprecipitation and binding assays.

• Regulatory mechanism investigation: Evidence suggests CABP4 affects Cav1.4 through structural interference with the binding of the inhibitor of Ca²⁺-dependent inactivation (ICDI) domain to the C-terminus of Cav1.4 . Antibodies can help map these regulatory interactions.

• Complex formation analysis: Research indicates that CaBP4 forms part of the Cav1.4 channel complex in the retina . Antibodies can help investigate the composition and stoichiometry of these channel complexes.

• Developmental regulation: CABP4 is essential for proper development and maintenance of photoreceptor synapses . Antibodies can track expression patterns throughout development to understand temporal regulation.

What methodological approaches are recommended for studying the G155D mutation in CABP4?

Based on published research on the CABP4 G155D mutation associated with ADNFLE, these methodological approaches are recommended:

• Combined mRNA and protein analysis:

  • Real-time PCR to quantify mRNA expression levels

  • Western blot with CABP4 antibodies to assess protein expression

  • This approach revealed that the G155D mutation increases mRNA expression while decreasing protein levels, suggesting reduced protein stability

• Cell transfection experiments:

  • Create recombinant plasmids (e.g., pEGFP-N1-CABP4 wild-type and pEGFP-N1-CABP4-p.G155D)

  • Transfect human neuronal cells

  • Compare cellular phenotypes between wild-type, mutant, and control groups

• Protein stability assessment:

  • Cycloheximide chase assays with antibody detection to measure protein half-life

  • Proteasome inhibition experiments to investigate degradation pathways

• Electrophysiological correlation:

  • Patch-clamp technology to measure action potentials in neurons expressing wild-type versus mutant CABP4

  • Correlate with antibody-based protein quantification

What are common challenges when using CABP4 antibodies and how can they be resolved?

When working with CABP4 antibodies, researchers may encounter these challenges:

• Low signal intensity:

  • Increase antibody concentration within recommended ranges

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection methods

  • Enrich samples through immunoprecipitation before analysis

• Non-specific binding:

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Increase washing duration and frequency

  • Verify antibody specificity using blocking peptides

  • Consider more specific monoclonal antibodies if available

• Inconsistent results between experiments:

  • Standardize sample preparation protocols

  • Use consistent positive controls across experiments

  • Prepare larger batches of working antibody dilutions

  • Document lot numbers as antibody performance may vary between lots

• Cross-reactivity with other calcium-binding proteins:

  • Select antibodies targeting unique regions of CABP4

  • Validate using knockout/knockdown controls

  • Perform peptide competition assays

How can researchers optimize immunofluorescence protocols for CABP4 detection in retinal tissues?

For optimal immunofluorescence detection of CABP4 in retinal tissue:

• Tissue preparation:

  • Use fresh-frozen or properly fixed retinal tissue (4% paraformaldehyde for 1-2 hours)

  • Consider antigen retrieval methods if necessary

  • Use thin sections (10-12 μm) for better antibody penetration

• Blocking and antibody conditions:

  • Block with normal serum matching the secondary antibody host

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization

  • Incubate with primary antibody overnight at 4°C

  • Select antibodies that have been validated for immunofluorescence applications

• Visualization strategies:

  • Use photoreceptor-specific markers (e.g., rhodopsin, cone opsins) for co-localization

  • Include synaptic markers to verify localization at photoreceptor terminals

  • Employ confocal microscopy for precise localization

• Controls:

  • Include peptide-blocked antibody controls

  • Compare with known CABP4 expression patterns

  • Use retinal tissue from CABP4-deficient animals as negative controls

How can CABP4 antibodies advance research into epilepsy mechanisms?

The recent discovery of CABP4's potential role in epilepsy opens new research directions:

• Neuronal excitability assessment:

  • Use CABP4 antibodies to correlate protein expression with electrophysiological properties

  • The G155D mutation in CABP4 has been shown to increase action potential frequency in hippocampal neurons, potentially contributing to ADNFLE

• Comparative expression studies:

  • Investigate CABP4 expression patterns in epilepsy-relevant brain regions versus retina

  • Compare calcium channel regulation mechanisms between these tissues

• Genetic screening correlation:

  • Support genetic findings with protein-level confirmation using antibodies

  • Help validate new mutations through functional studies combining antibody detection with electrophysiology

• Therapeutic target validation:

  • Screen compounds that might stabilize mutant CABP4 protein

  • Monitor treatment effects on CABP4 expression and localization

The p.G155D mutation was first discovered in a 4-generation pedigree with ADNFLE, representing a novel pathogenic mechanism that could be further explored through antibody-based approaches .

What methodological considerations are important when using CABP4 antibodies in high-throughput screening applications?

For adaptation to high-throughput screening contexts:

• Assay miniaturization:

  • Optimize antibody concentrations for microplate formats

  • Determine minimum cell numbers needed for reliable detection

  • Test detection sensitivity limits in automated systems

• Reproducibility considerations:

  • Standardize cell culture conditions for consistent CABP4 expression

  • Create stable cell lines expressing wild-type or mutant CABP4

  • Develop robust positive and negative controls

• Multiplexing strategies:

  • Combine CABP4 antibodies with markers for calcium channel proteins

  • Develop dual-detection systems for simultaneous assessment of protein levels and calcium signaling

  • Consider phosphorylation-specific antibodies if regulatory modifications are identified

• Data analysis approaches:

  • Establish quantitative thresholds for positive hits

  • Incorporate machine learning for pattern recognition in complex phenotypes

  • Validate screening hits with orthogonal antibody-based methods