GABRA6 Antibody

What is GABRA6 and why is it significant in neuroscience research?

GABRA6 (gamma-aminobutyric acid type A receptor subunit alpha6) is a critical component of GABAA receptors, which are ligand-gated chloride channels that mediate the major inhibitory neurotransmission in the mammalian brain. GABRA6 is a protein with 453 amino acid residues and a molecular mass of approximately 51 kDa in humans, although it often appears at around 72 kDa in gel electrophoresis due to post-translational modifications . It is predominantly expressed in the cerebellum and cerebral cortex, making it a valuable marker for studying inhibitory neurotransmission in these brain regions . The protein belongs to the ligand-gated ion channel (TC 1.A.9) protein family and participates in critical signal transduction pathways . Its significance in neuroscience research stems from its involvement in various neurological conditions, including childhood absence epilepsy associated with specific mutations such as R46W . Additionally, genetic variants like T1521C have been linked to personality characteristics and stress responses, making GABRA6 relevant for broader behavioral neuroscience investigations .

What are the recommended applications for GABRA6 antibodies?

GABRA6 antibodies have been validated for multiple research applications with Western Blot (WB) and ELISA being the most commonly employed techniques . For Western Blot applications, dilutions typically range from 1:500 to 1:2000, while ELISA applications may require more diluted preparations (approximately 1:40000) . Immunoprecipitation (IP) is another well-established application, particularly valuable for studying protein-protein interactions involving GABRA6 . Some antibodies, such as GABRA6-R25, have been additionally validated for immunohistochemistry (IHC) and immunofluorescence (IF), expanding their utility for tissue localization studies . When using anti-GABRA6 antibodies for Blue Native PAGE-antibody shift experiments, they can effectively detect GABRA6-containing protein complexes and reveal interactions with other subunits like α1 . For optimal results across all applications, researchers should verify the specific reaction conditions recommended for their particular antibody clone or formulation.

What species reactivity should be considered when selecting GABRA6 antibodies?

When selecting GABRA6 antibodies, researchers must carefully consider species reactivity to ensure compatibility with their experimental models. Available GABRA6 antibodies demonstrate varying cross-reactivity patterns. Some antibodies, like those described in search result , exhibit broad reactivity across human, mouse, and rat species, making them versatile tools for comparative studies . In contrast, other antibodies may have more limited reactivity profiles, such as the GABRA6-R25 antibody, which has been confirmed specifically for rat GABRA6 . This consideration is particularly important as GABRA6 gene orthologs have been identified in multiple species including mouse, rat, bovine, frog, chimpanzee, and chicken . For researchers working with less common model organisms, verification of antibody cross-reactivity may require additional validation experiments. When designing studies involving multiple species or using antibodies with limited documented cross-reactivity, preliminary testing with positive controls from the target species is strongly recommended to confirm specificity and appropriate binding characteristics.

Why is the observed molecular weight of GABRA6 often different from its calculated weight?

The discrepancy between the calculated molecular weight of GABRA6 (approximately 51 kDa) and its observed molecular weight in experimental conditions (often around 72 kDa) is a common source of confusion for researchers . This difference primarily stems from post-translational modifications of the protein, particularly glycosylation, which can significantly increase its apparent molecular weight on SDS-PAGE gels. GABRA6, as a membrane-bound receptor subunit, contains multiple glycosylation sites necessary for proper protein folding, trafficking, and function . Additionally, the hydrophobic nature of this transmembrane protein can affect its migration pattern in gel electrophoresis. Other factors that may contribute to the observed molecular weight difference include the use of different sample preparation methods, gel systems, or running conditions. When validating antibodies, researchers should be aware of this discrepancy and not dismiss antibodies that detect bands at higher molecular weights than theoretically predicted. Validation experiments, such as those shown in search result , often include peptide competition assays where the antibody is pre-incubated with its immunizing peptide to confirm specificity of the observed bands at the higher molecular weight.

How can researchers validate the specificity of GABRA6 antibodies?

Validating the specificity of GABRA6 antibodies is essential for ensuring reliable experimental results. Several complementary approaches are recommended. First, peptide competition assays should be performed wherein the antibody is pre-incubated with the synthetic peptide used as the immunogen. As demonstrated in western blot analyses from search result , specific bands should disappear or be significantly reduced in intensity when the antibody is blocked with the synthesized peptide . Second, researchers should test the antibody on known positive controls (tissues or cell lines with confirmed GABRA6 expression, such as cerebellum) and negative controls (tissues or cell lines lacking GABRA6 expression). Third, comparative analysis using multiple antibodies targeting different epitopes of GABRA6 can increase confidence in specificity. Additionally, validation by immunoprecipitation followed by mass spectrometry analysis, as described in search result , provides robust confirmation of antibody specificity . This approach can identify co-immunoprecipitating proteins and verify the presence of GABRA6. For genetic validation, researchers can use GABRA6 knockout or knockdown models where the target protein is absent or reduced. Lastly, cross-reactivity testing across species should be performed if the antibody will be used in multiple model organisms, as reactivity may vary significantly between species despite high sequence homology.

What experimental controls are essential when using GABRA6 antibodies?

When designing experiments with GABRA6 antibodies, incorporating appropriate controls is essential for generating reliable and interpretable data. Primary controls should include positive tissue controls, particularly cerebellum and cerebral cortex samples where GABRA6 is highly expressed . Negative controls should incorporate tissues known to lack GABRA6 expression or samples from GABRA6 knockout models when available. For antibody specificity validation, peptide competition assays should be performed, where the antibody is pre-incubated with the immunizing peptide before application to the sample . This should eliminate or significantly reduce specific binding. In immunoprecipitation experiments, researchers should include isotype-matched non-specific antibody controls to identify non-specific binding . For western blot applications, loading controls using housekeeping proteins (e.g., β-actin, GAPDH) are crucial to normalize for protein loading variations. When performing immunohistochemistry, antibody concentration must be carefully titrated, and both primary antibody omission and isotype controls should be included. For experiments investigating GABRA6 interactions with other subunits, such as the α1/α6-containing subcomplexes described in search result , controls should include antibodies against other GABA receptor subunits to confirm specificity of the observed interactions .

What are the optimal tissue preparation methods for GABRA6 immunodetection?

Optimal tissue preparation for GABRA6 immunodetection varies depending on the specific application, but several key considerations apply across techniques. For Western blot and immunoprecipitation applications, fresh or flash-frozen brain tissue, particularly from cerebellum and cerebral cortex where GABRA6 is predominantly expressed, should be homogenized in cold lysis buffers containing appropriate protease inhibitors to prevent protein degradation . Non-denaturing conditions may be preferred when studying GABRA6 in its native protein complexes, as used in Blue Native PAGE experiments described in search result . For immunohistochemistry and immunofluorescence, perfusion fixation with 4% paraformaldehyde followed by careful cryoprotection is generally recommended, though optimal fixation conditions may vary between antibodies. Antigen retrieval methods may be necessary to expose the GABRA6 epitope, particularly when the antibody targets regions that may be masked during fixation. When preparing membrane proteins like GABRA6, it's important to use detergents that effectively solubilize membrane components without disrupting antibody binding sites. For subcellular fractionation studies, protocols should be optimized to enrich for cell membrane fractions where GABRA6 is localized . Regardless of the preparation method, samples should be processed quickly and maintained at cold temperatures to minimize protein degradation and preserve GABRA6 immunoreactivity.

How can Blue Native PAGE-antibody shift techniques be used to study GABRA6-containing protein complexes?

Blue Native PAGE (BN-PAGE) coupled with antibody shift assays represents a powerful technique for investigating native GABRA6-containing protein complexes. This method preserves protein-protein interactions by separating protein complexes in their native state. In this approach, tissue extracts (typically cerebellum for GABRA6 studies) are treated with specific anti-GABRA6 antibodies prior to BN-PAGE separation . The antibody binding causes a shift in the electrophoretic mobility of GABRA6-containing complexes, which can be detected by subsequent immunoblotting with antibodies against GABRA6 or potential interaction partners . As demonstrated in search result , this technique successfully revealed that α1 and α6 subunits can be assembled into the same receptor complex, predominantly at a molecular weight of approximately 450 kDa . The method allows researchers to distinguish between different GABRA6-containing subcomplexes based on their molecular weight and composition. For example, the main population of α6-containing subcomplexes was found to migrate at 450 kDa and did not co-migrate with synaptic markers like Nlgn2 or Gphn . To implement this technique effectively, researchers must optimize antibody concentrations to achieve sufficient binding for observable shifts without causing precipitation of the complexes. The specificity of the observed shifts should be validated using control antibodies and confirmed through alternative approaches such as co-immunoprecipitation followed by mass spectrometry.

What methods are recommended for investigating GABRA6 subunit interactions with other GABA receptor components?

Investigation of GABRA6 interactions with other GABA receptor components requires multi-faceted methodological approaches. Immunoprecipitation (IP) coupled with mass spectrometry represents the gold standard for identifying protein interaction partners . As detailed in search result , anti-GABRA6 antibodies can effectively immunoprecipitate GABRA6-containing complexes, allowing for the identification of associated proteins through subsequent mass spectrometric analysis . This approach successfully revealed that the GABRA1 subunit is present in GABRA6-containing complexes . Complementary to this, Blue Native PAGE with antibody shift assays provides a powerful method to specifically confirm subunit compositions within native receptor complexes. This technique demonstrated that α1 and α6 subunits can assemble into the same receptor, particularly in complexes of approximately 450 kDa . For visualization of interactions in cellular contexts, proximity ligation assays (PLA) or Förster resonance energy transfer (FRET) can be employed using fluorescently labeled antibodies against GABRA6 and potential interaction partners. Crosslinking studies with bifunctional crosslinkers followed by immunoprecipitation and mass spectrometry can identify transient or weak interactions. Additionally, co-expression studies in heterologous systems, where GABRA6 and other subunits are expressed with different tags, provide a controlled environment to study assembly and trafficking of receptor complexes. Each of these methods offers distinct advantages, and combining multiple approaches provides the most comprehensive understanding of GABRA6 interactions.

What approaches are recommended for studying the functional effects of GABRA6 mutations?

Studying the functional effects of GABRA6 mutations, such as the R46W mutation associated with childhood absence epilepsy (CAE), requires a comprehensive experimental strategy . Electrophysiological approaches, particularly patch-clamp recording in heterologous expression systems, provide direct measurement of how mutations affect channel function, including changes in GABA sensitivity, channel kinetics, and conductance properties. These experiments can reveal how mutations like R46W reduce αβγ and αβδ receptor function, potentially leading to neuronal disinhibition and increased seizure susceptibility . Complementary to electrophysiology, surface expression assays using cell-surface biotinylation or immunofluorescence with non-permeabilized cells can determine whether mutations affect receptor trafficking to the plasma membrane. For in vivo investigations, CRISPR-Cas9 gene editing can be used to introduce specific mutations into animal models, allowing assessment of behavioral, electrophysiological, and biochemical consequences. Biochemical approaches, such as co-immunoprecipitation with antibodies against GABRA6 and its interaction partners, can reveal how mutations affect protein-protein interactions within the receptor complex . Structural biology techniques, including cryo-electron microscopy, may provide insights into how mutations alter receptor conformation. Finally, molecular dynamics simulations can predict how amino acid substitutions affect protein structure and function at the atomic level. By combining these diverse approaches, researchers can gain comprehensive understanding of how GABRA6 mutations contribute to pathophysiological conditions like childhood absence epilepsy.

How should researchers address discrepancies in GABRA6 antibody results across different experimental platforms?

When confronted with discrepancies in GABRA6 antibody results across different experimental platforms, researchers should implement a systematic troubleshooting approach. First, antibody specificity should be reassessed through peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific signals . Different antibody lots may exhibit batch-to-batch variations, necessitating lot-specific validation. Experimental conditions, including buffer composition, pH, detergent concentration, and incubation times, should be optimized for each platform independently, as conditions optimal for Western blot may not translate directly to immunohistochemistry or ELISA. Researchers should consider epitope accessibility, which may vary between applications due to differences in protein conformation or fixation effects. For membrane proteins like GABRA6, sample preparation methods critically influence results, particularly concerning membrane solubilization and protein denaturation . When possible, multiple antibodies targeting different GABRA6 epitopes should be employed to validate findings. Additionally, researchers should verify that the observed molecular weight corresponds to expectations, noting that GABRA6 often appears at approximately 72 kDa despite its calculated weight of 51 kDa due to post-translational modifications . Finally, experimental results should be interpreted within the context of known GABRA6 expression patterns, with highest expression expected in cerebellum and cerebral cortex . By systematically addressing these factors, researchers can resolve discrepancies and generate reliable, reproducible data across experimental platforms.

What quantitative approaches are recommended for analyzing GABRA6 expression levels?

Quantitative analysis of GABRA6 expression levels requires careful methodological considerations to ensure accuracy and reproducibility. For Western blot quantification, densitometric analysis should be performed using linear range exposures, with GABRA6 signals normalized to appropriate loading controls such as β-actin or GAPDH . Standard curves using recombinant GABRA6 protein can enhance quantitative precision. Quantitative PCR (qPCR) offers a complementary approach for measuring GABRA6 transcript levels, requiring careful primer design to ensure specificity and efficiency. ELISA assays, with recommended antibody dilutions of approximately 1:40000, provide another quantitative method, particularly valuable for high-throughput screening . For tissue-level quantification, stereological approaches in immunohistochemistry enable unbiased estimation of GABRA6-positive cell numbers and density. Flow cytometry can quantify GABRA6 surface expression in single-cell suspensions when using antibodies targeting extracellular epitopes. Mass spectrometry-based approaches, particularly selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), offer highly specific and sensitive protein quantification . For each quantitative method, appropriate statistical analyses should be employed, including tests for normal distribution, homogeneity of variance, and selection of parametric or non-parametric tests accordingly. Researchers should report both effect sizes and confidence intervals in addition to p-values. Multi-platform validation, combining several quantitative approaches, provides the most robust assessment of GABRA6 expression levels, particularly when investigating subtle changes in experimental or pathological conditions.