GNGT2 Antibody

What is GNGT2 and what cellular functions does it participate in?

GNGT2 (G protein subunit gamma transducin 2) is a critical component of G protein signaling pathways, playing crucial roles in mediating cellular responses to external stimuli. It belongs to the G protein gamma family and is specifically localized in cone photoreceptors . GNGT2 is particularly important in phototransduction processes within these cells. The protein is involved in various transmembrane signaling systems where G proteins function as modulators or transducers. The beta and gamma chains (including GNGT2) are required for GTPase activity, GDP-to-GTP replacement, and G protein-effector interactions . Multiple transcript variants encoding the same protein have been identified for this gene, indicating complex regulation of its expression .

What are the structural and biochemical characteristics of the GNGT2 protein?

GNGT2 is a relatively small protein with a calculated molecular weight of approximately 8 kDa, though it is often observed at about 13 kDa in Western blot analyses, suggesting post-translational modifications . The protein sequence consists of 69 amino acids in humans (NP_113686.1) . GNGT2 is typically localized to the cell membrane, specifically on the cytoplasmic side, and features a lipid anchor . The amino acid sequence of human GNGT2 is: MAQD LSEK DLLK MEVE QLKK EVKN TRIP ISKA GKEI KEYV EAQA GNDP FLKG IPED KNPF KEKG GCLI S . This sequence is important for recognizing appropriate antibody targets in research applications.

What are common synonyms for GNGT2 in scientific literature?

When researching GNGT2 in scientific databases, it's important to be aware of its multiple designations. Common synonyms include:

• GNG9

• HG3I

• GNGT8

• G-GAMMA-8

• G-GAMMA-9

• G-GAMMA-C

• Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-T2

• Transducin gamma subunit

• Guanine nucleotide regulatory protein gamma c subunit

Using these alternative names in literature searches ensures comprehensive coverage of relevant research.

What factors should be considered when selecting a GNGT2 antibody for specific applications?

Selection of appropriate GNGT2 antibodies requires careful consideration of multiple factors:

• Application compatibility: Verify the antibody has been validated for your intended application (WB, ELISA, IHC) .

• Species reactivity: Most commercially available GNGT2 antibodies react with human and mouse samples .

• Antibody type: Most GNGT2 antibodies are polyclonal, raised in rabbits, and target specific amino acid sequences of the protein .

• Immunogen information: Consider the specific sequence used as immunogen (commonly amino acids 1-69 of human GNGT2) .

• Detection method: Determine compatibility with your preferred detection system (fluorescent, colorimetric, etc.) .

• Observed vs. calculated MW: Be aware that GNGT2 runs at approximately 13 kDa on gels despite its calculated 8 kDa MW .

Understanding these factors ensures selection of an appropriate antibody that will yield reliable results for your specific experimental conditions.

What are the optimal protocols for Western blot analysis using GNGT2 antibodies?

For optimal Western blot results with GNGT2 antibodies, consider the following methodological approach:

• Sample preparation: Extract proteins from appropriate tissue (spleen is a positive control for mouse samples) .

• Protein loading: Use 20-25 μg of protein per lane .

• Recommended dilution: 1:500 to 1:2000 for primary antibody incubation .

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:10,000 dilution .

• Blocking buffer: 3% nonfat dry milk in TBST .

• Detection method: ECL Basic Kit with exposure times of approximately 90 seconds .

• Expected band size: Look for bands at approximately 13 kDa, despite the calculated MW of 8 kDa .

This protocol has been validated for detection of GNGT2 in mouse spleen samples and can be adapted for other tissues based on GNGT2 expression levels.

How should GNGT2 antibodies be stored to maintain optimal activity?

To preserve antibody activity and prevent degradation, GNGT2 antibodies should be stored according to the following recommendations:

• Storage temperature: Maintain at -20°C for long-term storage .

• Buffer composition: Typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: Divide into smaller volumes upon receipt to avoid repeated freeze-thaw cycles .

• Handling precautions: Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce sensitivity .

• Shipping conditions: Typically shipped with ice packs; store immediately upon receipt .

Following these storage guidelines ensures maximum stability and performance of GNGT2 antibodies throughout the research project timeline.

How are GNGT2 antibodies used in neuroscience research, particularly related to phototransduction?

GNGT2 antibodies are valuable tools in neuroscience research, particularly for studying phototransduction processes:

• Cone photoreceptor studies: GNGT2 is specifically localized in cone photoreceptors, making antibodies useful for distinguishing cone from rod photoreceptor populations .

• Phototransduction pathway analysis: GNGT2 antibodies help elucidate the regulation of signaling proteins in the visual transduction cascade .

• Cell-type specific signaling: These antibodies enable investigation of G protein signaling specifically in cone photoreceptors versus other retinal cell types .

• Retinal development studies: GNGT2 antibodies can track the expression and localization of this protein during retinal development .

• Visual system disease models: These antibodies help examine alterations in cone-specific signaling in various visual system pathologies .

These applications leverage the specificity of GNGT2 to cone photoreceptors, allowing researchers to investigate specialized aspects of visual signal processing and retinal function.

What is the relationship between GNGT2 and Alzheimer's disease based on recent research?

Recent research has revealed intriguing connections between GNGT2 and Alzheimer's disease (AD) pathology:

• Gene co-expression: Abi3 and Gngt2 genes show tight co-expression correlation in multiple AD patient cohorts, including temporal cortex samples (Mayo TCX: ρ = 0.644, p = 2.2e-16), cerebellar samples (Mayo CER: ρ = 0.556, p = 2.2e-16), and the Religious Orders Study and Rush Memory and Aging Project cohort (ROSMAP: ρ = 0.328, p = 2.2e-16) .

• Animal models: Co-regulation of Abi3 and Gngt2 has been confirmed in APP transgenic TgCRND8 mice (ρ = 0.625, p = 7.31-06) and MAPT transgenic rTg4510 mice (ρ = 0.554, p = 0.018) .

• Pathological effects: Deletion of Abi3-Gngt2 resulted in:

  • Gene dose- and age-dependent reduction in Aβ deposition in APP mice

  • Exacerbation of tauopathy and astrocytosis when pro-aggregant human tau was expressed

  • Upregulation of AD-associated immune gene expression profiles (including Trem2, Plcg2, and Tyrobp) even without AD neuropathology

• Inflammatory modulation: Abi3-Gngt2-/- mice show upregulation of immune pathways including granulocyte and leukocyte chemotaxis, proliferation of mononuclear leukocytes, and leukocyte-mediated immunity .

This research highlights the complex relationship between GNGT2 and AD, suggesting inflammatory gliosis can have opposing effects on amyloid and tau pathology.

What methodological approaches can be used to study GNGT2 phosphorylation and its functional implications?

Studying GNGT2 phosphorylation requires specialized techniques to detect and characterize post-translational modifications:

• Phospho-specific antibodies: Though not specifically mentioned in the search results, phospho-specific antibodies could be developed to target potential phosphorylation sites on GNGT2.

• Mutational analysis: The AD-associated S209F variant of ABI3 (which is co-expressed with GNGT2) alters phosphorylation patterns, suggesting similar approaches could be used for GNGT2 .

• Mass spectrometry: Protein samples immunoprecipitated with GNGT2 antibodies can be analyzed by mass spectrometry to identify and quantify phosphorylation sites.

• Functional assays: After identifying phosphorylation sites, site-directed mutagenesis can be used to create phosphomimetic or phosphodeficient mutants to study functional implications.

• Kinase inhibition studies: Treatment with various kinase inhibitors followed by Western blot analysis can help identify kinases responsible for GNGT2 phosphorylation.

These approaches can help elucidate how phosphorylation affects GNGT2 function in signal transduction pathways and potentially in pathological conditions.

What are common challenges in Western blot analysis with GNGT2 antibodies and how can they be addressed?

Researchers may encounter several challenges when using GNGT2 antibodies for Western blot analysis:

• Molecular weight discrepancy: GNGT2 shows a discrepancy between calculated (8 kDa) and observed (13 kDa) molecular weights .

  • Solution: Always verify positive controls and use protein markers spanning low molecular weights.

• Weak signal strength:

  • Solution: Optimize primary antibody concentration (try 1:500 instead of 1:2000), increase protein loading to 25 μg, extend primary antibody incubation time, or use enhanced chemiluminescence detection systems .

• Background issues:

  • Solution: Increase blocking time (1-2 hours), use 3-5% nonfat dry milk in TBST, add 0.005% SDS to secondary antibody dilution, and ensure thorough washing between steps .

• Sample preparation:

  • Solution: Use freshly prepared tissue samples when possible, include protease inhibitors during extraction, and verify protein concentration using BCA assay .

• Antibody specificity:

  • Solution: Include both positive and negative controls, use tissues known to express GNGT2 (like mouse spleen), and validate with knockout samples when available .

Systematic optimization of these parameters will improve detection of GNGT2 by Western blot.

How can immunohistochemical protocols be optimized for GNGT2 detection in tissue sections?

For optimal immunohistochemical detection of GNGT2 in tissue sections, consider the following methodological refinements:

• Tissue preparation: Use formalin-fixed paraffin-embedded sections with standard deparaffinization procedures, followed by antigen retrieval using steam .

• Blocking conditions: Block in 2% FBS in 1× PBS for 1 hour at room temperature to reduce non-specific binding .

• Antibody dilution: For IHC applications, use dilutions between 1:50-1:300, optimizing based on tissue type and expression levels .

• Incubation conditions: Incubate with primary antibody overnight at 4°C for optimal antigen binding .

• Detection system: Use appropriate secondary antibody systems such as ImmPress reagents followed by DAB detection and hematoxylin counterstaining .

• Positive control tissues: Human tonsil has been verified as a positive control for GNGT2 antibody in IHC applications .

• Counterstaining options: For co-localization studies, consider ThioS staining (1% ThioS for 7 min at room temperature) followed by brief washing in 70% ethanol and mounting with fluorescent mounting medium containing DAPI .

These optimizations enhance the specificity and sensitivity of GNGT2 detection in histological specimens.

How can GNGT2 antibodies contribute to understanding neurodegenerative disease mechanisms?

GNGT2 antibodies offer valuable insights into neurodegenerative disease mechanisms, particularly for Alzheimer's disease:

• Immune response modulation: Deletion of Abi3-Gngt2 upregulates immune gene expression profiles including Trem2, Plcg2, and Tyrobp that are associated with AD, suggesting GNGT2 antibodies can help monitor these changes .

• Pathology tracking: GNGT2 antibodies can be used to track how Abi3-Gngt2 expression correlates with changes in both amyloid and tau pathologies in age-dependent manners .

• Cell-type specific analyses: As microglia are the cell types most affected in Abi3-Gngt2-/- mice (p<0.05), GNGT2 antibodies can help identify microglial-specific changes in neurodegenerative conditions .

• Biomarker development: By understanding GNGT2 expression patterns in disease states, researchers may develop new diagnostic or prognostic biomarkers for neurodegenerative diseases.

• Therapeutic target validation: GNGT2 antibodies can help validate potential therapeutic targets by monitoring changes in protein expression following experimental interventions.

These applications demonstrate how GNGT2 antibodies contribute to our understanding of complex neurodegenerative disease mechanisms and potential therapeutic approaches.

What advanced experimental designs can incorporate GNGT2 antibodies for studying G protein signaling networks?

Researchers can implement sophisticated experimental designs using GNGT2 antibodies to investigate G protein signaling networks:

• Co-immunoprecipitation studies: Use GNGT2 antibodies to pull down protein complexes and identify novel interaction partners through mass spectrometry, helping map G protein signaling networks .

• CRISPR-Cas9 gene editing: Generate GNGT2 knockout or knock-in models (similar to the Abi3-Gngt2-/- mice), then use GNGT2 antibodies to confirm deletion and study downstream effects on signaling pathways .

• Proximity ligation assays: Combine GNGT2 antibodies with antibodies against potential interaction partners to visualize and quantify protein-protein interactions at subcellular resolution.

• Multiplexed immunofluorescence: Use GNGT2 antibodies in combination with other signaling protein markers to create comprehensive maps of G protein signaling in specific tissues or disease states.

• Live-cell imaging: Generate fluorescently tagged GNGT2 constructs and validate their localization and function using GNGT2 antibodies as a reference for the endogenous protein.

• Single-cell transcriptomic correlation: Correlate protein-level GNGT2 data from antibody-based studies with single-cell RNA-seq data to better understand cell-type specific functions and expression patterns.

These advanced approaches leverage GNGT2 antibodies to gain deeper insights into G protein signaling networks across different biological contexts.

What are the emerging research areas where GNGT2 antibodies may provide critical insights?

Several emerging research areas could benefit from GNGT2 antibody applications:

• Neuroinflammation in retinal diseases: Given GNGT2's specific expression in cone photoreceptors, antibodies can help investigate how cone-specific signaling changes contribute to retinal pathologies .

• Microglial activation signatures in neurodegeneration: GNGT2 antibodies can help characterize specific microglial activation states, such as the antiquewhite2 module identified in Abi3-Gngt2-/- mice, which contains genes (Ctss, Siglec h, Csf3r, Ly86, C1qc) reported in both mouse models and human AD cases .

• G protein signaling in immune modulation: Research connecting GNGT2 with immune pathways like granulocyte and leukocyte chemotaxis suggests potential roles in broader immune regulation that can be explored with antibodies .

• Aging-related changes in signal transduction: The age-dependent effects of Abi3-Gngt2 deletion on amyloid pathology suggest GNGT2 antibodies could help investigate how G protein signaling changes throughout the aging process .

• Drug discovery for neurodegenerative diseases: GNGT2 antibodies can facilitate high-throughput screening approaches to identify compounds that modulate G protein signaling with therapeutic potential .

These emerging areas represent opportunities where GNGT2 antibodies could provide valuable tools for advancing scientific understanding and developing new therapeutic approaches.