CACNB4 Antibody
Description
Introduction to CACNB4 and Its Antibody
CACNB4 is encoded by the CACNB4 gene and plays a pivotal role in calcium channel regulation, influencing neurotransmitter release, muscle contraction, and neuronal signaling. Mutations in this gene are linked to neurological disorders such as epilepsy and episodic ataxia . The CACNB4 antibody is used to detect this protein in various experimental systems, enabling insights into its subcellular localization and functional interactions.
Research Applications of CACNB4 Antibody
The antibody is validated for multiple techniques:
Dilution Guidelines:
Neurological Disorders
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Epilepsy and Ataxia: CACNB4 mutations disrupt calcium channel function, leading to neuronal hyperexcitability. Antibodies have identified aberrant β4 localization in epilepsy models .
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Schizophrenia: Overexpression of CACNB4 reduces dendritic spine density in cortical neurons, with sex-specific effects observed in female mice .
Cellular and Molecular Mechanisms
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Channel Modulation: CACNB4 interacts with α1 subunits (e.g., Cav2.1) to regulate calcium influx and synapse organization .
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Protein Interactions: IP studies using CACNB4 antibodies revealed associations with Bassoon and CAST/Erc2, critical for active zone assembly .
Clinical Relevance
Disease Associations:
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Epilepsy: Heterozygous CACNB4 mutations (e.g., p.Cys104Phe) linked to idiopathic generalized epilepsy .
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Episodic Ataxia: Mutations impair channel gating, causing cerebellar dysfunction .
Diagnostic Potential: Antibodies enable detection of β4 subunit mislocalization or expression changes in patient samples, aiding in disease diagnosis and mechanism studies .
Technical Considerations
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Antigen Retrieval: Required for IHC (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0) .
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Cross-Reactivity: Polyclonal antibodies may exhibit broader reactivity (e.g., rat, pig, rabbit) , while monoclonal antibodies show higher specificity .
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Controls: Positive controls include mouse/rat brain lysates or transfected cell lines .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The nuclear targeting properties of the truncated beta(4b(1-481)) subunit were investigated in tsA-201 cells, skeletal myotubes, and hippocampal neurons. PMID: 24875574
- Genome-wide association studies have identified CACNB4 mutations linked to juvenile myoclonic epilepsy. PMID: 23756480
- Cacnb4 directly couples electrical activity to gene expression, a process that is defective in juvenile epilepsy. PMID: 22892567
- The Ca2+ channel beta4c subunit interacts with heterochromatin protein 1 gama via a PXVXL binding motif. PMID: 21220418
- CACNB4 is associated with acute lung injury in mice. PMID: 21297076
- The novel nucleotide substitution T87C (D29D) in CACNB4 was observed in 2 migrainous vertigo patients and was not present in control DNA samples. PMID: 16866717
- No pathogenic mutations were identified in CACNB4. PMID: 18446307
- CACNB4 plays a role in neurotransmitter release. PMID: 18712068
Q&A
How should researchers validate CACNB4 antibody specificity for Western blot applications?
Validation requires a multi-step approach:
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Knockout controls: Use tissue lysates from CACNB4 knockout models to confirm absence of nonspecific bands .
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Cross-reactivity checks: Compare reactivity across species (e.g., human vs. mouse) using antibodies with known epitopes, such as the C-terminal-targeting clone Everest Biotech #EB06591 .
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Blocking peptide assays: Pre-incubate antibodies with immunizing peptides (e.g., proprietary C-terminal peptide for ABIN2854649 ) to verify signal loss.
Example validation data:
| Antibody Clone | Observed Band (kDa) | Knockout Validation | Cross-Reactivity (Species) |
|---|---|---|---|
| Everest Biotech #EB06591 | 58-60 | Confirmed | Human, Mouse |
| Proteintech 17770-1-AP | 58 | Mouse brain | Human, Mouse, Rat |
What factors influence antibody performance in immunohistochemistry (IHC) for CACNB4?
Key methodological considerations:
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Antigen retrieval: Use TE buffer (pH 9.0) for formalin-fixed tissues to expose CACNB4 epitopes .
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Sex-specific signal quantification: In cortical tissue, normalize signal intensity to β1b levels due to sex-dependent interactome differences .
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Negative controls: Include no-primary-antibody and IgG-isotype controls to rule out background in brain sections .
How do researchers select antibodies for co-immunoprecipitation (co-IP) studies of CACNB4 interactomes?
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Epitope location: Use C-terminal-targeting antibodies (e.g., ABIN2854649 ) to avoid disrupting N-terminal binding domains critical for α1 subunit interactions .
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Buffer compatibility: Optimize lysis buffers to preserve β4-β1b interactions, which are male-enriched and stabilize complexes .
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Validation: Confirm pulldown specificity via mass spectrometry (as in ) and reciprocal IPs with β1b antibodies.
How do sex differences impact CACNB4 antibody-based spine density measurements?
Experimental design requirements:
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Stratify by sex: Female C57BL/6J mice show 23% greater β4-mediated small spine loss versus males .
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Estrous cycle controls: Despite estrous-independent effects , track cycle phases via vaginal cytology to exclude hormonal confounders.
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Interactome analysis: Combine IP with quantitative proteomics to detect male-specific β1b enrichment (Fig. 3 in ).
Key findings:
| Sex | Small Spine Density (spines/μm) | β1b/β4 Interaction Strength |
|---|---|---|
| Male | 1.14 ± 0.09 | 3.2-fold higher |
| Female | 0.87 ± 0.11* | Baseline |
| *p < 0.01 vs. male, n = 10/group |
What methodologies resolve contradictions between in vitro and in vivo CACNB4 overexpression phenotypes?
Case study: β4OE reduces small spines in vivo (female mice) but not consistently in vitro .
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System-specific factors:
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Resolution strategy:
How should researchers interpret CACNB4 missense mutations (e.g., C104F) in functional studies?
Methodological framework:
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Electrophysiological profiling: Express mutants in HEK293T cells with CaV2.1 subunits and quantify inactivation kinetics (τ_fast) .
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Structural modeling: Map C104F to the β4 SH3 domain to predict disrupted α1 binding .
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In vivo complementation: Introduce mutants into Cacnb4⁻/⁻ mice and assess seizure thresholds .
Functional data:
What controls are essential when using CACNB4 antibodies in schizophrenia-related spine loss models?
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Postmortem interval (PMI) controls: Match PMI ≤ 12 hrs to prevent epitope degradation .
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Subcellular fractionation: Isolate synaptic membranes to concentrate β4-β1b complexes .
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Longitudinal design: Track spine dynamics from neurodevelopment (P14) to adulthood (P84) .
