GNAO1 Antibody

What is GNAO1 and what cellular functions does it regulate?

GNAO1 is the alpha subunit of heterotrimeric guanine nucleotide-binding protein (G protein), a membrane protein widely expressed in the central nervous system. It functions as a molecular switch in signal transduction pathways by alternating between active (GTP-bound) and inactive (GDP-bound) states. The protein contains a guanine nucleotide binding site and possesses a low GTPase activity that converts bound GTP to GDP, thereby terminating signals .

GNAO1 primarily inhibits adenylate cyclase activity, which leads to decreased intracellular cAMP levels . Recent research has demonstrated that GNAO1 also plays a critical role in regulating Rho signaling pathways that control cytoskeletal remodeling and neurite outgrowth, suggesting its importance in neuronal morphology and connectivity .

Where is GNAO1 primarily expressed and what is its molecular weight?

GNAO1 is predominantly expressed in brain tissue, particularly in neurons. It has been detected in mouse and rat brain tissues, as well as human neural tissues. The calculated molecular weight of GNAO1 is approximately 35 kDa (302 amino acids), though the observed molecular weight in experimental conditions is typically around 40 kDa as determined by Western blot analysis . Expression has also been detected in other tissues including mouse testis, rat testis, human liver, and Y79 cells (a retinoblastoma cell line) .

What criteria should be considered when selecting a GNAO1 antibody for research?

When selecting a GNAO1 antibody, researchers should consider:

• Intended application: Different antibodies perform optimally in specific applications. For example, the rabbit polyclonal antibody ab154001 is suitable for Western blot (WB), immunocytochemistry/immunofluorescence (ICC/IF), and immunohistochemistry (IHC-P) , while antibody 12635-1-AP is validated for WB, IHC, immunofluorescence (IF), immunoprecipitation (IP), and ELISA applications .

• Species reactivity: Verify the antibody's reactivity with your experimental model. For instance, 12635-1-AP shows reactivity with human, mouse, and rat samples .

• Immunogen information: The epitope recognized by the antibody affects specificity. For example, ab154001 targets a recombinant fragment within human GNAO1 (amino acids 100-350) , while 12635-1-AP is raised against a GNAO1 fusion protein .

• Validation data: Review available validation data, including published citations, to ensure the antibody's reliability for your specific application.

• Clonality and host species: Consider whether a polyclonal or monoclonal antibody best suits your needs, and ensure the host species is compatible with your experimental design.

How should GNAO1 antibodies be validated before experimental use?

A comprehensive validation approach for GNAO1 antibodies should include:

• Positive and negative controls: Use tissues known to express GNAO1 (e.g., brain tissue) as positive controls and tissues with minimal expression as negative controls.

• Knockdown/knockout verification: Utilize siRNA-mediated knockdown of GNAO1 (as demonstrated in Neuro2a cells with >90% reduction in expression) to confirm antibody specificity .

• Western blot analysis: Verify single band detection at the expected molecular weight (approximately 40 kDa) .

• Cross-reactivity assessment: Test for potential cross-reactivity with other G protein alpha subunits, particularly closely related family members.

• Application-specific validation: For immunohistochemistry, compare staining patterns with established neuroanatomical distribution of GNAO1. For immunoprecipitation, confirm pull-down specificity through mass spectrometry.

What are the optimal conditions for using GNAO1 antibodies in Western blotting?

For optimal results, researchers should include reducing agents in sample buffers and ensure complete denaturation of membrane proteins. The higher observed molecular weight (40 kDa) versus calculated (35 kDa) may be due to post-translational modifications, which should be considered when interpreting results .

How should GNAO1 antibodies be applied in immunohistochemistry for neural tissues?

For successful immunohistochemical detection of GNAO1 in neural tissues:

• Tissue fixation and processing: Formalin-fixed, paraffin-embedded sections work well, though freshly prepared samples yield optimal results.

• Antigen retrieval: Use TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0 as an alternative .

• Antibody dilution: Use at 1:50-1:500 dilution, optimizing for each specific antibody and tissue .

• Detection system: Either fluorescent or enzymatic (e.g., HRP-DAB) detection systems are suitable.

• Controls: Include brain tissue sections as positive controls and sections treated with secondary antibody only as negative controls.

• Counterstaining: Hematoxylin for nuclear visualization with DAB detection, or DAPI for fluorescent applications.

GNAO1 typically shows cytoplasmic and membranous staining in neurons, with particularly strong expression in certain brain regions. When interpreting results, consider that altered localization has been correlated with specific phenotypes, such as developmental and epileptic encephalopathy .

How can GNAO1 antibodies help elucidate the molecular mechanisms of GNAO1-related disorders?

GNAO1 antibodies are instrumental in investigating the molecular pathophysiology of GNAO1-related disorders through several advanced applications:

• Subcellular localization studies: Using immunofluorescence with GNAO1 antibodies helps determine if pathogenic variants affect protein localization. Research has shown that decreased localization of Gαo in the plasma membrane correlates with developmental and epileptic encephalopathy 17 .

• Protein-protein interaction analysis: Immunoprecipitation with GNAO1 antibodies followed by mass spectrometry can identify binding partners affected by pathogenic variants. This approach has revealed that molecular defects caused by pathogenic Gαo include an inability to interact with cellular binding partners .

• Functional pathway investigation: Combined with RNA sequencing data, GNAO1 antibodies help verify protein-level changes in signaling pathways. For example, siRNA-mediated depletion of GNAO1 perturbs expression of genes associated with Rho signaling, validating a role for GNAO1 in cytoskeletal remodeling essential for neurite outgrowth .

• Genotype-phenotype correlation studies: Immunohistochemical and biochemical analyses have established correlations between specific variants and clinical phenotypes. Variants in position 203 relate to developmental and epileptic encephalopathy, while those in position 209 associate with neurodevelopmental disorder with involuntary movements .

What methodological approaches are recommended for studying GNAO1's role in neuronal differentiation and neurite outgrowth?

To investigate GNAO1's role in neuronal differentiation and neurite outgrowth, researchers should consider these methodological approaches:

• Knockdown studies: siRNA-mediated depletion of GNAO1 (>90% knockdown efficiency) in neuronal cell lines like Neuro2a provides a powerful model to assess morphological and functional consequences .

• Neurite outgrowth quantification: Following GNAO1 knockdown or antibody-based interventions, analyze neurite length and number using immunofluorescence with cytoskeletal markers (e.g., F-actin with phalloidin staining and tubulin β3 with TUJ1 antibodies) .

• Subcellular analysis: Dissect neurite structures into stem and tip regions to precisely quantify immunofluorescence signals of cytoskeletal components and their relationship to GNAO1 expression .

• Nuclear-cytoplasmic ratio assessment: Measure the nuclear-cytoplasmic (NC) ratio as an indicator of cellular state, as GNAO1-depleted cells exhibit higher NC ratios (0.486 vs. 0.416 in controls) .

• Transcriptomic profiling: Combine with RNA sequencing to identify differentially expressed genes in GNAO1-depleted cells, particularly focusing on Rho signaling pathway components .

What are common challenges in GNAO1 antibody experiments and how can they be addressed?

How can researchers optimize detection of GNAO1 in different neural cell types and developmental stages?

Optimizing GNAO1 detection across neural cell types and developmental stages requires:

• Developmental timing: GNAO1 expression varies during development. For embryonic studies, use specialized fixation protocols (e.g., 4% PFA for shorter duration) and adjust antibody concentrations accordingly.

• Cell type-specific considerations:

  • For neurons: Co-staining with neuronal markers like MAP2 or NeuN helps identify GNAO1 expression patterns in specific neuronal populations.

  • For glial cells: Though primarily neuronal, low-level GNAO1 expression in glia may require signal amplification techniques.

• Tissue-specific optimization: Brain regions differ in GNAO1 expression levels. Higher antibody concentrations may be needed for regions with lower expression.

• Subcellular localization: Use confocal microscopy with membrane markers to precisely localize GNAO1, especially important as membrane localization correlates with functional status .

• Signal amplification: For developmental stages with lower expression, consider tyramide signal amplification or similar techniques to enhance detection sensitivity.