G0S2 Antibody

What is G0S2 and why is it important in research?

G0S2 is a small basic protein of 103 amino acids that was originally identified in blood mononuclear cells during cell cycle progression. While initially believed to mediate re-entry of cells from G0 to G1 phase of the cell cycle, its best-characterized function is as a direct inhibitor of adipose triglyceride lipase (ATGL), a rate-limiting enzyme in lipolysis . G0S2 has gained research importance due to its multifunctional roles in diverse cellular processes including lipid metabolism, cellular proliferation, differentiation, and apoptosis . Recent studies have also implicated G0S2 in controlling endothelial cell proliferation through specific signaling pathways .

What types of G0S2 antibodies are available for research?

Researchers can access several types of G0S2 antibodies, including rabbit monoclonal antibodies like the ZooMAb® Anti-G0S2 Antibody (clone 2C13) expressed in HEK 293 cells and rabbit polyclonal antibodies generated against purified, recombinant His-tagged G0S2 produced in E. coli . These antibodies have been validated for various applications including Western blotting, immunohistochemistry on paraffin-embedded tissues, and immunofluorescence .

What are the validated applications for G0S2 antibodies?

G0S2 antibodies have been validated across multiple experimental applications:

How does G0S2 protein subcellular localization affect antibody selection?

G0S2 exhibits multiple subcellular localizations that can influence antibody selection. Immunofluorescence staining has revealed G0S2 localization at the surface of lipid droplets , while other studies demonstrate mitochondrial localization where it interacts with Bcl-2 . Additionally, G0S2 can be found in the cytosol where it interacts with nucleolin . When selecting an antibody, researchers should consider which subcellular pool of G0S2 they aim to detect and whether the antibody has been validated for that specific localization pattern. For comprehensive studies, using multiple antibodies targeting different epitopes can help distinguish between G0S2's various functional pools.

How can I differentiate between G0S2's lipid metabolism and apoptotic functions in my experiments?

Distinguishing between G0S2's dual roles in lipid metabolism and apoptosis requires careful experimental design:

• Subcellular fractionation approach: Isolate mitochondrial fractions (where G0S2 interacts with Bcl-2 for apoptotic functions) and lipid droplet fractions (where G0S2 inhibits ATGL) separately, then perform Western blotting with the G0S2 antibody on both fractions .

• Co-immunoprecipitation strategy: Use G0S2 antibodies to co-immunoprecipitate different binding partners - pull-down experiments showing G0S2-ATGL interaction indicate lipid metabolism functions, while G0S2-Bcl-2 interactions suggest apoptotic pathway involvement .

• Functional readouts: Measure triglyceride accumulation, fatty acid release, and fatty acid oxidation to assess lipid metabolism effects , while simultaneously monitoring apoptotic markers (caspase activation, PARP cleavage) after G0S2 manipulation.

• Mutagenesis approach: Utilize targeted mutations in the hydrophobic domain of G0S2 (required for ATGL interaction) versus mutations in regions required for Bcl-2 binding to selectively disrupt specific functions while preserving others .

What are the best experimental controls when manipulating G0S2 expression levels?

When manipulating G0S2 expression through overexpression or knockdown experiments, consider these critical controls:

For G0S2 overexpression studies:

• Use GFP-expressing adenovirus as a negative control, as demonstrated in human primary myotube studies

• Include a non-functional G0S2 mutant (with mutations in the hydrophobic domain) to confirm specificity of observed effects

• Measure G0S2 protein levels by Western blot to confirm overexpression magnitude (typically 4-5 fold increases are achievable)

• Monitor unexpected compensatory changes in ATGL expression, as G0S2 overexpression has been shown to increase ATGL protein content by 21% in some systems

For G0S2 knockdown experiments:

• Employ scramble shRNA (shNT) controls alongside G0S2-targeting shRNA

• Verify knockdown efficiency at both mRNA (>60% reduction) and protein levels (near-complete loss is achievable)

• Monitor cell viability between control and knockdown conditions

• Perform rescue experiments by reintroducing shRNA-resistant G0S2 expression constructs to confirm phenotype specificity

• Consider double knockdown experiments (G0S2 with interacting partners like ATGL) to establish pathway dependencies

How can I resolve contradictory G0S2 expression data between different tissue types?

Researchers frequently encounter seemingly contradictory G0S2 expression patterns across different tissues and cellular contexts. To resolve these discrepancies:

• Normalize to appropriate reference genes: The stability of reference genes varies between tissues; use multiple reference genes validated for each specific tissue type.

• Consider tissue-specific isoforms: Verify that your G0S2 antibody recognizes all relevant isoforms in your tissues of interest.

• Account for oxidative vs. glycolytic metabolism: G0S2 expression is higher in oxidative muscles (like soleus) compared to glycolytic muscles (gastrocnemius and extensor digitorum longus) , suggesting metabolic profile strongly influences expression levels.

• Examine fiber type composition: Within skeletal muscle, G0S2 may be preferentially expressed in type I oxidative fibers , so differences in fiber type composition between samples can explain apparent contradictions.

• Consider regulatory context: G0S2 expression can be regulated by PPARs, cell cycle state, and inflammatory signals , necessitating careful documentation of the physiological and metabolic state of each tissue sample.

What methodological approaches can address G0S2's dual roles in cell proliferation?

G0S2 displays seemingly contradictory roles in cell proliferation—both promoting cell cycle re-entry in some contexts while maintaining quiescence in others, particularly in hematopoietic stem cells . To investigate these context-dependent functions:

• Cell type-specific knockdown and overexpression: Compare G0S2 manipulation effects across proliferative vs. quiescent cell populations.

• Nucleolin interaction studies: As G0S2 directly interacts with nucleolin affecting cell proliferation, perform co-localization experiments using G0S2 antibodies together with nucleolin antibodies across different cell cycle stages .

• ChIP-seq approaches: Identify G0S2-dependent changes in chromatin accessibility during cell cycle transitions.

• Single-cell analysis: Correlate G0S2 protein levels (via immunofluorescence) with cell cycle markers in heterogeneous populations to capture cell-to-cell variation.

• Functional readouts: Combine BrdU incorporation, Ki-67 staining, and cell cycle phase analysis when assessing G0S2's impact on proliferation.

What are the optimal extraction conditions for detecting G0S2 in different sample types?

G0S2 detection requires specific extraction approaches depending on the sample type:

For tissue samples:

• For whole tissue lysates, homogenization in 1X Laemmli buffer has been successfully used for G0S2 detection by Western blot

• For lipid-rich tissues, consider using methods that preserve lipid droplet integrity while allowing protein extraction

• For preserving phosphorylation states of G0S2, include phosphatase inhibitors in extraction buffers

For cultured cells:

• Direct lysis in Laemmli buffer is effective for total G0S2 detection

• For subcellular fractionation, gentle cell disruption methods followed by differential centrifugation are recommended to separate mitochondrial, cytosolic, and lipid droplet fractions

• For co-immunoprecipitation studies, non-denaturing lysis buffers that preserve protein-protein interactions should be used

How can I optimize immunohistochemical detection of G0S2 in different tissues?

Successful immunohistochemical detection of G0S2 requires:

• Antigen retrieval optimization: For formalin-fixed, paraffin-embedded tissues, TE buffer (pH 9.0) is recommended for G0S2 antibodies, with citrate buffer (pH 6.0) as an alternative .

• Antibody titration: Begin with a dilution range of 1:50-1:500 and optimize based on signal-to-noise ratio .

• Positive control selection: Use tissues known to express high levels of G0S2, such as mouse liver tissue or rat heart tissue, which have been validated for positive staining .

• Signal amplification considerations: For tissues with lower G0S2 expression, consider using polymer-based detection systems or tyramide signal amplification.

• Counterstaining optimization: Adjust hematoxylin counterstaining intensity to ensure G0S2 signal remains visible while maintaining nuclear morphology for contextual analysis.

What strategies can address the challenges of studying G0S2-ATGL interactions?

The G0S2-ATGL interaction presents unique experimental challenges. Here are methodological approaches to address them:

• In vitro ATGL activity assays: Recombinant human G0S2 (rhG0S2) can be used to test ATGL inhibition under different conditions, including in the presence of CGI-58 co-activator .

• Competition binding assays: Determine whether G0S2 competes with CGI-58 for ATGL binding or if they bind through different mechanisms.

• Domain mapping approaches: Utilize the knowledge that the hydrophobic domain of G0S2 interacts with the patatin-like domain of ATGL to create domain-specific mutations for interaction studies.

• Functional lipolysis assays: Compare effects of G0S2 manipulation on both basal and hormone-stimulated lipolysis in adipocytes and tissue explants .

• Microscopy approaches: Co-localization studies at lipid droplet surfaces can provide spatial information about the G0S2-ATGL interaction using appropriate antibodies for both proteins.

How should researchers interpret changes in G0S2 expression in disease models?

When analyzing G0S2 expression changes in disease models, consider these methodological aspects:

• Expression analysis methods: Compare results from multiple techniques including qPCR, Western blotting with G0S2 antibodies, and immunohistochemistry to get a comprehensive view of expression changes.

• Timing considerations: Temporal aspects of G0S2 expression are critical—in response to proliferative activation, G0S2 mRNA expression peaks between 1-2 hours .

• Survival correlation analysis: In cancer studies such as uveal melanoma, perform Kaplan-Meier survival analysis comparing high versus low G0S2 expression groups (defined by median expression values) .

• Pathway context: Consider G0S2 expression changes within the context of specific pathways—for example, in autoimmune conditions like vasculitis, correlate G0S2 upregulation with autoimmunity-related markers such as anti-nuclear antibody and anti-double strand DNA antibody levels .

• Heterogeneous biological network analysis: For complex diseases, integrate G0S2 expression data into heterogeneous biological networks that connect chemical, gene, disease, pathway, and phenotype nodes to identify relevant interactions .

How can I troubleshoot weak or non-specific G0S2 antibody signals?

When encountering issues with G0S2 antibody signals, consider these problem-solving approaches:

• Antibody validation: Confirm antibody specificity using G0S2 knockdown or knockout samples as negative controls.

• Sample preparation optimization: For Western blotting, test different protein extraction methods and buffer compositions; G0S2 (being a lipid droplet-associated protein) may require specialized extraction protocols.

• Signal enhancement strategies:

  • For Western blotting, increase protein loading (up to 50-100 μg), extend primary antibody incubation time (overnight at 4°C), or use more sensitive detection substrates

  • For immunohistochemistry, optimize antigen retrieval conditions and consider amplification systems

  • For immunofluorescence, use brighter fluorophores or signal amplification methods

• Background reduction:

  • Increase blocking reagent concentration (5% BSA or milk)

  • Test different blocking agents (normal serum matched to secondary antibody species)

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffers for better penetration

  • Extend washing steps (5x 5 minutes) with gentle agitation

What are the critical factors affecting reproducibility in G0S2 functional studies?

Reproducibility in G0S2 functional studies depends on several critical factors:

• Expression system selection: The choice between adenoviral, lentiviral, or plasmid-based expression systems significantly impacts G0S2 expression levels and cellular responses .

• Knockdown efficiency verification: Always confirm G0S2 knockdown at both mRNA (>60% reduction) and protein levels (near-complete loss is achievable) .

• Cell type considerations: G0S2 functions differently across cell types—effects seen in adipocytes may differ from those in myotubes, hepatocytes, or hematopoietic cells .

• Metabolic state standardization: Standardize the metabolic state of cells (fed vs. fasted, insulin-stimulated vs. basal) as these conditions significantly affect G0S2 expression and function.

• Interaction partner expression levels: Document expression levels of key G0S2 interaction partners (ATGL, Bcl-2, nucleolin) as their relative abundance affects G0S2 function .

How can G0S2 antibodies be applied in cancer metabolism research?

G0S2 antibodies offer valuable tools for investigating cancer metabolism through several approaches:

• Cancer subtype stratification: G0S2 expression analysis in cancer samples can help stratify tumors, as demonstrated in uveal melanoma where G0S2 expression correlates with patient outcomes .

• Metabolic reprogramming assessment: G0S2's role in lipid metabolism makes it a key marker for investigating metabolic shifts in cancer cells from glucose to lipid metabolism.

• Therapeutic response monitoring: Track G0S2 expression and localization in response to metabolic-targeting therapeutics using immunohistochemistry on patient-derived xenografts or clinical samples.

• Dual-function investigation: Investigate how G0S2's dual roles in lipid metabolism and apoptosis (via Bcl-2 interaction) may be exploited or dysregulated in cancer cells .

• Cell cycle regulation: Examine G0S2's potential role in cancer stem cell quiescence through its interaction with nucleolin and effects on cell cycle regulation .

What novel methodological approaches could enhance G0S2 protein interaction studies?

Emerging methodologies offer new possibilities for studying G0S2 interactions:

• Proximity labeling approaches: BioID or APEX2 fusion constructs with G0S2 can identify proximal proteins in living cells, potentially revealing new interaction partners beyond the known ATGL, Bcl-2, and nucleolin interactions.

• Single-molecule imaging: Techniques like PALM or STORM microscopy using fluorescently tagged G0S2 can reveal the dynamics of G0S2 localization and interactions at nanoscale resolution.

• Integrative omics approaches: Combine G0S2 antibody-based proteomics (immunoprecipitation-mass spectrometry) with transcriptomics and lipidomics for comprehensive pathway analysis.

• Graph convolutional networks: Apply advanced computational methods as described in search result to integrate G0S2 into larger heterogeneous biological networks connecting chemicals, genes, diseases, pathways, and phenotypes.

• Structural biology approaches: While challenging due to G0S2's size and properties, structural studies of G0S2-partner complexes could provide mechanistic insights into its inhibitory functions.