CACNG6 Antibody

What is CACNG6 and what is its role in calcium channel function?

CACNG6 encodes the calcium channel voltage-dependent gamma subunit 6, which is an integral membrane protein that stabilizes calcium channels in their inactive (closed) state. It plays a regulatory role in voltage-dependent calcium channels, particularly L-type calcium channels containing CACNA1C as the pore-forming subunit . Calcium channel gamma subunits comprise eight members that share a common topology, with CACNG6 belonging to the first cluster along with gamma-1 . These channels regulate calcium influx essential for several physiological processes including synaptic transmission, muscle contraction, neurogenesis, hormone secretion, cell motility, division, and development .

What is the structure and localization pattern of CACNG6?

CACNG6 protein structure includes four transmembrane domains with short intracellular N- and C-termini. The first transmembrane domain contains a specific GxxxA motif that is critical for its inhibitory function . The protein is primarily expressed in striated muscle cells, with significant presence in skeletal and cardiac muscles. A distinct isoform is also expressed at lower levels in the brain . Immunohistochemical staining using anti-CACNG6 antibodies has shown expression in the pyramidal layer of the rat cingulate cortex .

How do I select the appropriate CACNG6 antibody for my research?

Selection should be based on:

• Target epitope: Different antibodies target various regions of CACNG6:

  • N-terminal region antibodies (e.g., ABIN2776283)

  • Extracellular antibodies targeting amino acids 87-100 (e.g., ACC-112)

  • Middle region antibodies (amino acids 50-150)

• Species reactivity: Consider cross-reactivity with your experimental model:

  Antibody Example	Human	Mouse	Rat	Other Species
  ABIN2776283	100%	92%	92%	Dog (91%), Cow (82%), Guinea Pig (83%), Rabbit (93%)
  ACC-112	Yes	Yes	Yes	Not specified
  ab220833	Yes	Not tested	Not tested	Not specified

• Application compatibility: Confirm the antibody has been validated for your specific application (WB, IF, IHC) .

What are the optimal protocols for Western blotting using CACNG6 antibodies?

For Western blotting with CACNG6 antibodies:

• Sample preparation: Use cell or tissue lysates from appropriate sources (skeletal muscle, cardiac tissue, or neuronal samples based on your research question).

• Protein loading: CACNG6 has a molecular weight of approximately 24 kDa , so use appropriate gel percentage (12-15% SDS-PAGE).

• Antibody dilution: Typically 1:200 to 1:500 for primary antibody incubation, though optimal dilutions should be determined experimentally .

• Validation controls: Include positive controls (rat skeletal muscle lysate has been validated ) and negative controls (antibody preincubated with the immunizing peptide) .

• Detection: Standard secondary antibody and chemiluminescence or fluorescence-based detection systems are compatible.

A critical validation step is performing a blocking peptide experiment, where the antibody is preincubated with the immunizing peptide before application to the membrane, which should eliminate specific binding .

How can I optimize immunohistochemistry (IHC) protocols for CACNG6 detection?

Based on successful IHC experiments reported in the literature:

• Fixation: Use paraformaldehyde (PFA) fixation for optimal epitope preservation .

• Permeabilization: Triton X-100 has been successfully used for permeabilizing cells for CACNG6 staining .

• Antibody concentration: For immunohistochemical staining of rat brain tissue, a dilution of 1:400 has been effectively used with ACC-112 antibody .

• Visualization: CACNG6 can be visualized using fluorescently-labeled secondary antibodies (green fluorescence has been used successfully) .

• Counterstaining: DAPI counterstaining helps identify nuclear positioning relative to CACNG6 expression .

• Controls: Include both positive tissue controls (brain cingulate cortex for neuronal studies) and negative controls (antibody omission or preabsorption with immunizing peptide) .

What considerations should I take for immunofluorescence using CACNG6 antibodies?

For optimal immunofluorescence results:

• Cell preparation: For cultured cells such as A549, PFA fixation followed by Triton X-100 permeabilization has yielded good results .

• Antibody concentration: A concentration of 4 μg/mL has been reported effective for staining CACNG6 in A549 cells .

• Signal amplification: May be necessary for tissues with low CACNG6 expression; tyramide signal amplification can be considered.

• Co-localization studies: CACNG6 can be co-stained with calcium channel alpha subunits to study their interaction and localization.

• Imaging parameters: Use confocal microscopy for precise subcellular localization, particularly for membrane proteins like CACNG6.

How can CACNG6 antibodies be used to investigate associations with disease states?

CACNG6 polymorphisms have been linked to aspirin-intolerant asthma (AIA), making it an important research target. Antibodies can be used to:

• Expression profiling: Compare CACNG6 protein levels in tissues from patients with AIA versus controls using quantitative immunoblotting or immunohistochemistry.

• Variant-specific detection: Develop or select antibodies that can distinguish between protein variants encoded by different polymorphisms, such as the significant rs192808C>T variant (OR = 2.88, P = 0.0004) .

• Functional studies: Use antibodies to investigate how CACNG6 variants affect calcium channel function in airway cells, which may contribute to aspirin sensitivity.

• Tissue distribution analysis: Examine whether CACNG6 expression patterns differ in upper and lower airways between AIA patients and controls.

Research has shown significant association between CACNG6 SNPs and AIA:

These statistical associations can guide research into functional consequences that may be investigated using CACNG6 antibodies .

What approaches can be used to study CACNG6 interactions with other calcium channel components?

To investigate the regulatory role of CACNG6 within calcium channel complexes:

• Co-immunoprecipitation: Use CACNG6 antibodies to pull down the protein and associated channel components, followed by immunoblotting for interaction partners, particularly CACNA1C .

• Proximity ligation assay: Employ antibodies against CACNG6 and other channel subunits to visualize protein-protein interactions in situ with subcellular resolution.

• Sequential immunoprecipitation: For complex multi-protein assemblies, use sequential immunoprecipitation with different antibodies to isolate specific subcomplexes.

• Crosslinking studies: Combine chemical crosslinking with immunoprecipitation using CACNG6 antibodies to capture transient interactions.

• Functional modulation: Use antibodies targeting extracellular domains (like ACC-112 targeting amino acids 87-100) to modulate channel function in electrophysiological studies.

How can I validate the specificity of my CACNG6 antibody?

Rigorous validation is essential for reliable results with CACNG6 antibodies:

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide before application to your sample. This should eliminate specific staining, as demonstrated with ACC-112 antibody .

• Genetic validation: Use tissues or cells with CACNG6 knockdown or knockout as negative controls.

• Overexpression systems: Test antibody specificity in systems overexpressing CACNG6 compared to empty vector controls.

• Multiple antibody verification: Use antibodies targeting different epitopes of CACNG6 and compare staining patterns.

• Western blot molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of 24 kDa .

• Cross-reactivity testing: If working with multiple species, verify species cross-reactivity experimentally rather than relying solely on predicted reactivity percentages .

What are the key factors affecting CACNG6 antibody performance in different applications?

Several factors influence antibody performance:

• Epitope accessibility: The location of the epitope affects antibody binding in different applications:

  • N-terminal antibodies may work better for denatured proteins in Western blots

  • Extracellular antibodies are suitable for live cell applications and may access native conformations better

  • Conformation-dependent epitopes may be lost in fixed samples

• Fixation effects: Different fixatives can affect epitope preservation:

  • Paraformaldehyde works well for immunofluorescence of CACNG6

  • Methanol fixation may better preserve some epitopes but can disrupt membrane architecture

• Buffer composition: Sodium azide is used as a preservative in some CACNG6 antibody preparations (e.g., ABIN2776283)

• Storage and handling:

  • Store at -20°C for long-term storage

  • For short-term use (up to 1 week), 2-8°C is acceptable

  • Avoid repeated freeze-thaw cycles

How do I design experiments to investigate CACNG6 function using antibodies?

To effectively study CACNG6 function:

• Localization studies: Use immunofluorescence to determine subcellular localization in different cell types:

  • Membrane localization suggests active involvement in channel complexes

  • Intracellular retention might indicate regulatory mechanisms

• Expression correlation: Combine CACNG6 antibody staining with functional calcium imaging to correlate expression levels with channel activity.

• Manipulation experiments: Use function-blocking antibodies against extracellular domains to disrupt CACNG6 activity in live cells.

• Tissue-specific analysis: Compare CACNG6 expression and localization across tissues with differential calcium channel activity:

  • Skeletal muscle (high expression)

  • Cardiac tissue (high expression)

  • Brain regions (specific isoform with lower expression)

• Developmental studies: Track CACNG6 expression during development to understand its role in tissue maturation.

What techniques can be combined with CACNG6 antibodies for comprehensive functional studies?

Integrating multiple approaches provides deeper insights:

• Electrophysiology with immunostaining: Correlate calcium channel currents with CACNG6 expression levels in the same cells.

• FRET/BRET analysis: Use fluorescently labeled antibodies or tagged constructs to study dynamic interactions between CACNG6 and other channel components.

• Super-resolution microscopy: Employ techniques like STORM or PALM with CACNG6 antibodies to visualize nanoscale organization of calcium channel complexes.

• Calcium imaging: Combine calcium indicators with CACNG6 immunostaining to correlate protein expression with functional calcium signals.

• Single-cell transcriptomics with protein detection: Correlate CACNG6 mRNA expression with protein levels in individual cells to understand expression regulation.

• Mass spectrometry following immunoprecipitation: Identify novel interaction partners of CACNG6 by combining antibody-based pulldown with proteomic analysis.

How can I address common challenges when working with CACNG6 antibodies?

Researchers frequently encounter these issues:

• High background:

  • Solution: Optimize antibody concentration - test dilutions from 1:100 to 1:1000

  • Increase blocking time and concentration (5% BSA or normal serum from secondary antibody species)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific membrane binding

• No signal detection:

  • Confirm target expression in your sample (CACNG6 is highest in skeletal and cardiac muscle)

  • Try epitope retrieval methods for fixed tissues (citrate buffer, pH 6.0)

  • Verify antibody reactivity with your species (see predicted reactivity table)

• Multiple bands in Western blot:

  • CACNG6 is approximately 24 kDa ; bands at other sizes may represent:

    • Post-translational modifications

    • Splice variants

    • Non-specific binding

  • Increase washing stringency with higher salt concentration or detergent

• Inconsistent results:

  • Standardize protocols with positive controls (skeletal muscle)

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Use freshly prepared samples when possible

What are the implications of CACNG6 polymorphisms for antibody selection and experimental design?

The significant association of CACNG6 polymorphisms with disease states has implications for antibody-based research:

• Epitope considerations: Polymorphisms may affect antibody binding if they occur within the epitope region. For studies of polymorphic variants:

  • Select antibodies targeting conserved regions unaffected by known polymorphisms

  • Consider developing variant-specific antibodies for differential detection

• Population considerations: Studies involving rs192808C>T and other polymorphisms associated with AIA in Korean populations should consider:

  • Population-specific expression patterns

  • Genotyping subjects before protein expression analysis

  • Correlating antibody-based protein detection with genotype

• Functional relevance: Design experiments to correlate polymorphism-dependent changes in:

  • Protein expression levels

  • Subcellular localization

  • Interaction with other calcium channel components

  • Channel regulatory function

• Haplotype awareness: Consider complete haplotypes (e.g., CACNG6_BL1_ht6) rather than individual SNPs when interpreting antibody-based protein analysis.