CACNG8 Antibody

What is the molecular structure and function of CACNG8 protein?

CACNG8 is a voltage-dependent calcium channel gamma-8 subunit with a calculated molecular weight of approximately 43 kDa . It functions as a transmembrane AMPA receptor regulatory protein (TARP γ8) that modulates AMPA receptor activity in the central nervous system .

The protein plays crucial roles in regulating various aspects of AMPA receptors, including:

• Surface trafficking and expression

• Enhancement of synaptic clustering

• Increasing glutamate affinity

• Regulating channel properties and localization in the brain

CACNG8 belongs to the Type I TARP subfamily, which comprises four calcium channel γ subunits (γ2, γ3, γ4, and γ-8, also known as Cacng2, 3, 4, and 8, respectively) . As a non-pore-forming integral membrane protein with four transmembrane domains, it is predominantly expressed in the telencephalon with abundant distribution on hippocampal excitatory synapses and extrasynaptic membranes .

How should researchers select appropriate CACNG8 antibodies for specific experimental applications?

Selection should be guided by intended application, species reactivity, and epitope location. From the search results, available antibodies have been validated for different applications:

For applications requiring high specificity such as flow cytometry, monoclonal antibodies like ZRB1523 may be preferable, while polyclonal antibodies might provide stronger signals for IHC applications .

What factors influence antibody validation for CACNG8 detection in neuronal tissues?

Successful validation requires multiple complementary approaches:

• Specificity testing: Confirmation using blocking peptides to verify signal elimination, as demonstrated with ACC-125 antibody in rat brain lysates .

• Cross-species reactivity assessment: Different CACNG8 antibodies show variable reactivity across species. For example, 55078-1-AP reacts with human and mouse samples , while A10890 demonstrates reactivity with human, mouse, and rat samples .

• Multiple application validation: Comprehensive validation across different techniques ensures reliability. For instance, ZRB1523 has been validated for flow cytometry, immunocytochemistry, and immunohistochemistry .

• Proper control selection: Using brain tissues with known CACNG8 expression (e.g., hippocampus) alongside tissues with low expression as controls .

What are the optimal conditions for immunohistochemical detection of CACNG8 in brain tissues?

For successful IHC detection of CACNG8 in brain tissues, researchers should consider these protocol parameters:

• Antigen retrieval: For antibody 55078-1-AP, TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can serve as an alternative .

• Dilution optimization: Recommended dilutions vary by antibody:

  • 55078-1-AP: 1:50-1:500 for IHC

  • ZRB1523: 1:100 dilution successfully detected CACNG8/TARP in rat cerebral cortex tissue sections

• Detection method selection: Fluorescence-based detection allows visualization of CACNG8 in neuronal soma and processes, as demonstrated with ACC-125 antibody in rat hippocampus, where TARP γ8 staining appears in neuronal soma and neuronal processes .

• Counterstaining: Nuclear staining with DAPI provides context to CACNG8 localization patterns .

What are the recommended protocols for Western blot analysis of CACNG8 expression?

Based on available data, researchers should consider:

• Sample preparation: Brain tissue (particularly hippocampus) provides robust signal . Rat brain and mouse brain lysates have been successfully used to detect CACNG8 .

• Dilution optimization:

  • A10890: 1:500-2000 for Western blot applications

  • ACC-125: 1:400 dilution has been validated for rat and mouse brain lysates

• Controls: Include a blocking peptide control to confirm specificity, as demonstrated with ACC-125 antibody and its corresponding blocking peptide (BLP-CC125) .

• Expected molecular weight: Prepare to detect bands at approximately 43 kDa, which is the calculated molecular weight of CACNG8 .

How can CACNG8 antibodies be optimized for ELISA applications?

For ELISA applications, the following guidelines apply:

• Antibody selection: Multiple antibodies including 55078-1-AP and A10890 have been validated for ELISA applications.

• Dilution ranges:

  • For A10890, recommended ELISA dilutions range from 1:5000 to 1:20000

  • Titration is advised to determine optimal concentration for each experimental system

• Cross-reactivity considerations: Choose antibodies with validated reactivity to your species of interest. For example, A10890 demonstrates reactivity with human, mouse, and rat samples in ELISA applications .

How can CACNG8 antibodies contribute to understanding AMPA receptor trafficking mechanisms?

CACNG8 antibodies serve as valuable tools for investigating AMPA receptor regulation mechanisms:

• Co-localization studies: Antibodies enable visualization of CACNG8 distribution on hippocampal excitatory synapses and extrasynaptic membranes , providing insight into spatial relationships with AMPA receptors.

• Developmental expression analysis: CACNG8 expression differs temporally, with low levels in newborn and neonatal brain and higher levels in adult brain . Antibodies can track these developmental changes.

• Functional domain mapping: Different antibodies targeting specific epitopes (e.g., N-terminal regions like amino acids 2-17 ) help elucidate structure-function relationships in TARP γ8-mediated AMPA receptor regulation.

• Mechanistic investigations: CACNG8/TARP γ8 plays important roles in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines . Antibody-based approaches can help quantify these effects across different experimental conditions.

What methodologies can resolve contradictory findings when studying CACNG8 using different antibodies?

When facing contradictory results, researchers should implement these approaches:

• Epitope mapping comparison: Antibodies targeting different regions may yield different results. Compare epitope targets (e.g., ACC-125 targets N-terminus residues 2-17 while A10890 targets residues 140-220 ).

• Validation across multiple detection methods: Confirm findings using complementary techniques. For example, if IHC and Western blot results conflict, consider validation with additional methods like flow cytometry .

• Species-specific considerations: CACNG8 sequence variations across species may affect antibody binding. ZRB1523 shows direct reactivity to human and rat, with predicted reactivity to mouse, bovine, feline, and monkey based on sequence homology .

• Antibody format comparison: Compare results from polyclonal antibodies (e.g., 55078-1-AP , A10890 ) with monoclonal antibodies (e.g., ZRB1523 ) to identify potential epitope-specific effects.

How can researchers design experiments to investigate CACNG8's role in neurological disorders?

Experimental designs should incorporate:

• Regional expression analysis: Since γ-8 TARP is predominantly expressed in the telencephalon with abundant distribution on hippocampal excitatory synapses , targeted investigations of these regions in disease models may reveal pathological alterations.

• Developmental timing considerations: CACNG8 expression increases from newborn to adult brain , suggesting potential developmental vulnerability windows for disorders affecting glutamatergic transmission.

• Functional correlation studies: Combine antibody-based protein detection with electrophysiological measurements to correlate CACNG8 expression levels with AMPA receptor function in disease models.

• Therapeutic target validation: Use antibodies to evaluate CACNG8 as a potential therapeutic target, given its role in regulating expression, channel properties, and localization of AMPA receptors .

What strategies can address non-specific binding in CACNG8 antibody applications?

To minimize non-specific binding:

• Blocking optimization: Use appropriate blocking agents compatible with the host species of the primary antibody.

• Antibody titration: Determine optimal concentration through careful titration:

  • For 55078-1-AP: 1:50-1:500 for IHC

  • For A10890: 1:500-2000 for WB, 1:5000-20000 for ELISA

  • For ZRB1523: 1:100 for IHC and ICC

• Specificity controls: Incorporate blocking peptide controls where available (e.g., BLP-CC125 for ACC-125 antibody) to confirm signal specificity.

• Sample preparation optimization: For brain tissues, proper fixation and antigen retrieval (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0 ) significantly impact specific binding.

How should researchers interpret unexpected molecular weight variations in CACNG8 Western blots?

When observed molecular weight differs from the calculated ~43 kDa :

• Post-translational modifications: Consider potential glycosylation, phosphorylation, or other modifications that may alter migration patterns.

• Splice variant detection: Multiple isoforms may exist with different molecular weights.

• Sample preparation effects: Denaturing conditions, reducing agents, and buffer compositions can affect protein migration.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other TARP family members (γ2, γ3, γ4) which share structural similarities with γ8 .

• Degradation products: Partial degradation during sample preparation may generate lower molecular weight bands.