SUCLG2 Antibody

What is SUCLG2 and why is it important in biomedical research?

SUCLG2 is the GDP-specific beta subunit of succinyl-CoA synthetase, a critical enzyme in the tricarboxylic acid (TCA) cycle that catalyzes the conversion of succinyl-CoA to succinate while generating GTP. Recent studies have demonstrated that SUCLG2 is significantly overexpressed in lung adenocarcinoma (LUAD) compared to normal bronchial epithelial cells and is closely related to poor patient survival outcomes . Unlike SUCLA2 (the ATP-specific beta subunit), which shows inconsistent expression patterns in cancer, SUCLG2 exhibits consistent upregulation in tumor tissues, making it a particularly important target for cancer research . SUCLG2 has been shown to be essential for the proliferation of LUAD cells, as its knockout significantly inhibits tumor cell growth both in vitro and in vivo xenograft models without affecting normal bronchial epithelial cells . Furthermore, SUCLG2 plays a crucial role in maintaining mitochondrial function, regulating protein succinylation, and affecting the stability and activity of key metabolic enzymes, positioning it as a potential therapeutic target for cancer treatment.

How does SUCLG2 function differ from SUCLA2 in experimental systems?

While both SUCLG2 and SUCLA2 are beta subunits of succinyl-CoA synthetase, they exhibit distinct functional properties and expression patterns that are important to distinguish in experimental systems. SUCLG2 utilizes GDP as a substrate (GDP-specific), whereas SUCLA2 is ATP-specific . In lung adenocarcinoma research models, SUCLG2 shows consistent upregulation across cancer cell lines and tissues, while SUCLA2 exhibits variable expression with no consistent pattern . Knockout experiments reveal that SUCLG2 depletion, but not SUCLA2 knockout, significantly inhibits LUAD cell proliferation, indicating functionally divergent roles . This differential impact demonstrates that these two subunits are not functionally redundant despite catalyzing similar reactions. In experimental design, researchers should note that SUCLG2's effects on mitochondrial function appear more pronounced, as its knockout decreases mitochondrial DNA levels, increases reactive oxygen species production, decreases ATP content, and reduces mitochondrial membrane potential . These distinctions are critical when selecting which protein to target in cancer metabolism studies and when interpreting antibody-based detection results.

What are the known post-translational modifications of SUCLG2 relevant to antibody detection?

Post-translational modifications (PTMs) of SUCLG2 significantly impact its function and stability, with important implications for antibody detection methods. The most significant identified PTM is succinylation, particularly at the Lys93 residue, which has been shown to enhance SUCLG2 protein stability in LUAD cells . This modification creates a potential epitope that certain antibodies may detect differently than unsuccinylated SUCLG2. Additionally, SIRT5-mediated desuccinylation of SUCLG2 at Lys93 promotes its subsequent ubiquitination through K63-linkage by the E3 ubiquitin ligase TRIM21, leading to lysosomal degradation . When designing experiments, researchers should consider whether their antibodies can detect both succinylated and unsuccinylated forms, particularly if studying regulation of SUCLG2 stability. For comprehensive analysis, using antibodies targeting different epitopes or employing specialized antibodies that specifically recognize succinylated lysine may provide complementary information. Western blotting protocols may need optimization to preserve PTMs during sample preparation, including the use of deacetylase/desuccinylase inhibitors to prevent artificial removal of these modifications during experimental procedures.

What criteria should researchers use when selecting a SUCLG2 antibody for specific applications?

When selecting a SUCLG2 antibody, researchers should consider multiple parameters based on their specific experimental goals. First, evaluate the intended application—different antibodies are optimized for Western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF) . For instance, antibodies targeting amino acids 161-292 of SUCLG2 appear suitable for multiple applications including WB, IHC, and IF . Second, consider species reactivity—available antibodies show varying cross-reactivity with human, mouse, rat, and other species . For comparative studies across species, select antibodies with demonstrated multi-species reactivity, such as those recognizing the full-length protein (AA 1-384) . Third, evaluate the epitope location—antibodies targeting different regions of SUCLG2 may yield different results, especially if studying post-translational modifications like succinylation at Lys93 . Fourth, consider antibody format—unconjugated antibodies offer flexibility, while directly conjugated versions (HRP, biotin, FITC) may be advantageous for specific applications . Finally, prioritize knockout-validated antibodies for highest specificity confirmation, as these have been tested against samples lacking SUCLG2 expression . This multi-dimensional assessment ensures selection of the most appropriate antibody for generating reliable and reproducible results.

How can researchers validate SUCLG2 antibody specificity in their experimental systems?

Rigorous validation of SUCLG2 antibody specificity is crucial for experimental reliability and reproducibility. A comprehensive validation approach should employ multiple complementary strategies. First, utilize CRISPR-Cas9 knockout controls—researchers can generate SUCLG2 knockout cell lines similar to those described in published studies and confirm absence of signal with the selected antibody . Second, perform siRNA-mediated knockdown as an alternative validation method, which should result in proportionally decreased signal intensity . Third, implement peptide competition assays by pre-incubating the antibody with excess recombinant SUCLG2 protein or the immunizing peptide, which should abolish specific binding. Fourth, assess cross-reactivity by testing the antibody against related proteins, particularly SUCLA2, to ensure specificity within the succinyl-CoA synthetase family . Fifth, compare multiple antibodies targeting different epitopes of SUCLG2—concordant results across antibodies increase confidence in specificity. Finally, correlate protein detection with mRNA expression data where available. Document all validation procedures meticulously, including antibody catalog numbers, dilutions, incubation conditions, and imaging parameters to ensure reproducibility. This multi-faceted approach provides robust evidence for antibody specificity before proceeding with experimental applications.

What experimental controls are essential when using SUCLG2 antibodies in cancer research?

When employing SUCLG2 antibodies in cancer research, implementing comprehensive controls is essential for generating reliable and interpretable results. First, include positive controls such as cell lines known to express high levels of SUCLG2 (e.g., A549, H1299, HCC827 lung adenocarcinoma cells) alongside negative or low-expression controls like BEAS-2B normal bronchial epithelial cells . Second, incorporate genetic knockout or knockdown controls—CRISPR-Cas9-generated SUCLG2 knockout cells or siRNA-treated samples provide critical specificity validation . Third, when examining clinical samples, always include paired normal adjacent tissues for comparative analysis, as demonstrated in published LUAD tissue array studies . Fourth, for mechanistic studies involving succinylation, include controls treated with SIRT5 modulators to alter SUCLG2 post-translational modification status . Fifth, when assessing mitochondrial function in relation to SUCLG2, include mitochondrial markers (e.g., MitoTracker) to confirm co-localization and relevant functional controls for ATP production, ROS generation, and membrane potential . Finally, for quantitative analyses, implement technical replicates (minimum triplicate) and biological replicates across different cell lines or patient samples to ensure reproducibility. This comprehensive control strategy ensures that observed effects are specifically attributable to SUCLG2 and not experimental artifacts.

What are the optimal protocols for using SUCLG2 antibodies in Western blotting?

For optimal Western blotting of SUCLG2, researchers should implement a carefully optimized protocol that accounts for the protein's mitochondrial localization and post-translational modifications. Sample preparation begins with efficient extraction using mitochondrial isolation buffers containing protease inhibitors and, critically, desuccinylase inhibitors (e.g., nicotinamide) to preserve the succinylation status of SUCLG2 . Proteins should be separated on 10-12% SDS-PAGE gels, with expected migration of SUCLG2 at approximately 47 kDa. Transfer to PVDF membranes is recommended over nitrocellulose due to better protein retention. For blocking, 5% non-fat milk in TBST is generally effective, though BSA may provide lower background for some antibodies. Primary SUCLG2 antibody dilutions typically range from 1:1000 to 1:2000, with overnight incubation at 4°C showing optimal results . After thorough washing (4-5 times with TBST), apply species-appropriate HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution. For enhanced detection sensitivity, particularly when examining endogenous SUCLG2 in normal tissues with lower expression, ECL-Plus or similar enhanced chemiluminescence systems are recommended. Include size markers and positive controls (e.g., A549 cell lysate) alongside experimental samples . For quantification, normalize SUCLG2 signals to mitochondrial loading controls such as VDAC1 rather than cytosolic markers like GAPDH to account for potential variations in mitochondrial content between samples.

How should researchers optimize immunohistochemistry protocols for SUCLG2 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for SUCLG2 detection in tissue samples requires careful attention to multiple parameters to ensure specific and reproducible staining. Begin with appropriate tissue fixation—10% neutral buffered formalin for 24-48 hours is generally suitable, though excessive fixation can mask epitopes. Perform antigen retrieval using citrate buffer (pH 6.0) at 95-98°C for 20 minutes, as this has proven effective for revealing SUCLG2 epitopes in paraffin-embedded tissues . Blocking endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes followed by protein blocking with 5-10% normal serum is essential to reduce background. For primary antibody incubation, validated SUCLG2 antibodies should be applied at optimized dilutions (typically 1:100 to 1:500) and incubated overnight at 4°C for maximal sensitivity . Detection systems utilizing polymer-HRP technologies generally provide superior signal-to-noise ratios compared to conventional ABC methods. Counterstain with hematoxylin for 1-2 minutes to avoid obscuring specific SUCLG2 staining. For evaluation, implement standardized scoring systems as used in published LUAD tissue arrays, where both staining intensity and percentage of positive cells are considered . Always include positive controls (LUAD tissues) and negative controls (antibody diluent alone) on each slide. For dual-marker studies, consider using antibodies against relevant mitochondrial proteins or proliferation markers like Ki67, which has shown correlation with SUCLG2 expression in tumor samples .

How can SUCLG2 antibodies be employed to investigate mitochondrial dysfunction in cancer?

SUCLG2 antibodies serve as powerful tools for investigating the relationship between SUCLG2 and mitochondrial dysfunction in cancer through multiple advanced applications. Immunofluorescence co-localization studies using SUCLG2 antibodies alongside mitochondrial markers can reveal alterations in SUCLG2 distribution within the mitochondrial network in cancer cells compared to normal cells . Researchers can combine this approach with functional mitochondrial stains (MitoTracker, TMRE) to correlate SUCLG2 expression with membrane potential changes . For investigating molecular mechanisms, co-immunoprecipitation experiments using SUCLG2 antibodies can identify interaction partners such as SIRT5 and TRIM21, which regulate SUCLG2 stability through desuccinylation and ubiquitination, respectively . Proximity ligation assays offer higher sensitivity for detecting these protein-protein interactions in situ. Chromatin immunoprecipitation (ChIP) approaches utilizing antibodies against transcription factors that regulate SUCLG2 expression can elucidate how its overexpression is maintained in cancer cells. For translational applications, tissue microarray analyses with SUCLG2 antibodies can stratify patient samples based on expression levels, with published data showing significant correlation between high SUCLG2 expression and poor survival in LUAD patients (P = 0.0252) . This approach can be extended to assess whether SUCLG2 expression correlates with markers of mitochondrial function and patient response to therapies targeting mitochondrial metabolism.

What approaches can elucidate the relationship between SUCLG2 and protein succinylation networks?

Investigating the relationship between SUCLG2 and protein succinylation networks requires sophisticated methodological approaches that leverage specific antibodies and advanced techniques. Researchers should implement global succinylome analysis through mass spectrometry, comparing wild-type and SUCLG2 knockout or knockdown cells . This approach has revealed that SUCLG2 depletion increases succinylation of numerous mitochondrial proteins, with 61% of differentially succinylated proteins localized to mitochondria . To validate mass spectrometry findings for specific target proteins (e.g., GAPDH, ME2, IDH2, MDH2, ACOT9), use a sequential immunoprecipitation approach: first IP with antibodies against the target protein, then probe with anti-succinyl-lysine antibodies . For mechanistic studies, employ enzymatic activity assays of these metabolic enzymes in parallel with succinylation detection to establish cause-effect relationships between succinylation status and functional impairment . Site-directed mutagenesis of key lysine residues identified by mass spectrometry (e.g., K157 in ACOT9, K26 in ME2) to arginine can confirm the functional significance of specific succinylation sites . Time-course experiments using SUCLG2 inducible systems combined with succinylation detection can reveal the dynamics of succinylation changes. Finally, computational network analysis of the succinylome dataset can identify enriched pathways and protein clusters affected by SUCLG2-mediated changes in succinyl-CoA metabolism, providing insights into broader cellular reprogramming events beyond individual protein modifications .

How can researchers integrate SUCLG2 antibody-based detection with metabolomic analyses?

Integrating SUCLG2 antibody-based detection with metabolomic analyses provides powerful insights into the functional consequences of SUCLG2 expression alterations. Researchers should implement a multi-omics approach beginning with precise quantification of SUCLG2 protein levels using validated antibodies in western blotting or immunoprecipitation, followed by targeted metabolite analysis focusing on TCA cycle intermediates, particularly succinate and succinyl-CoA . Although direct measurement of succinyl-CoA is challenging due to oxidative hydrolysis, researchers can quantify succinic acid levels as a proxy for SUCLG2 activity, as demonstrated in studies showing reduced succinic acid content in SUCLG2 knockout cells . Stable isotope tracing experiments using 13C-labeled glucose or glutamine can track carbon flux through pathways influenced by SUCLG2, with samples collected at multiple time points and analyzed by GC-MS or LC-MS. For spatial resolution, combine metabolite imaging techniques with SUCLG2 immunofluorescence on sequential tissue sections to correlate metabolite distributions with protein expression patterns. To understand regulatory feedback loops, measure succinyl-CoA:succinate ratios in cellular compartments alongside SUCLG2 protein levels and succinylation status of target proteins. For clinical applications, correlate SUCLG2 antibody staining intensity in patient tissues with metabolomic profiles from the same samples to identify potential biomarkers or therapeutic vulnerabilities. This integrated approach reveals not only SUCLG2's impact on immediate substrate-product relationships but also broader metabolic reprogramming events in cancer cells that could be exploited therapeutically .

What are common pitfalls in SUCLG2 antibody-based experiments and how can they be addressed?

Researchers frequently encounter several technical challenges when working with SUCLG2 antibodies that require specific troubleshooting approaches. First, non-specific bands in Western blotting—this issue can be addressed by titrating antibody concentrations (starting with higher dilutions like 1:2000), using gradient gels to improve protein separation, and including knockout controls to identify the specific SUCLG2 band . Second, weak or absent signals—researchers should optimize extraction methods specifically for mitochondrial proteins, as standard whole-cell lysates may dilute the mitochondrial fraction where SUCLG2 is primarily localized . Third, inconsistent immunoprecipitation results—using crosslinking approaches and specialized lysis buffers containing desuccinylase inhibitors can preserve SUCLG2 interactions and post-translational modifications . Fourth, variable immunohistochemistry staining—standardize fixation times (24-48 hours in 10% neutral buffered formalin), optimize antigen retrieval conditions (citrate buffer pH 6.0, 20 minutes), and use positive control tissues with known SUCLG2 expression . Fifth, antibody cross-reactivity with SUCLA2—always validate with parallel experiments using SUCLA2 and SUCLG2 knockout models, as these related proteins share some sequence homology . Sixth, batch-to-batch variation—record lot numbers, prepare large stocks of working dilutions, and include internal standards across experiments. Finally, for quantitative analyses, establish standard curves using recombinant SUCLG2 protein to ensure measurements fall within the linear range of detection.

How should researchers interpret contradictory results when using different SUCLG2 antibodies?

When confronted with contradictory results from different SUCLG2 antibodies, researchers should implement a systematic analytical approach to resolve discrepancies. First, compare the epitope recognition regions of each antibody—antibodies targeting different domains of SUCLG2 may yield varying results, particularly if post-translational modifications or protein interactions mask certain epitopes . For instance, antibodies targeting the region containing Lys93 may show differential binding depending on the succinylation status of this residue . Second, evaluate the validation status of each antibody—prioritize results from knockout-validated antibodies over those without such rigorous validation . Third, consider application-specific optimization—antibodies optimized for Western blotting may not perform equivalently in immunohistochemistry or immunofluorescence applications . Fourth, assess species-specific differences—antibodies with different species reactivity profiles may perform inconsistently in cross-species studies . Fifth, examine experimental conditions—variations in fixation, sample preparation, or detection methods can differentially impact antibody performance. To resolve contradictions, implement orthogonal detection methods such as mass spectrometry or mRNA analysis to provide antibody-independent confirmation. Additionally, contact antibody manufacturers for technical support regarding known issues with specific lots or applications. Finally, report discrepancies in publications to improve community knowledge about antibody reliability, including detailed methodology that allows others to reproduce your findings.

What methodological adaptations are needed when studying SUCLG2 in different tissue and cell types?

Studying SUCLG2 across diverse tissue and cell types requires methodological adaptations to account for biological variations and technical challenges specific to each system. For cell lines with high mitochondrial content (e.g., muscle cells, neurons, cardiac cells), reduce lysate concentration in Western blotting to avoid signal saturation and use shorter exposure times . Conversely, for cells with lower mitochondrial content, enrich mitochondrial fractions before analysis to improve detection sensitivity. When working with primary tissues, adjust homogenization protocols—fibrous tissues (muscle, lung) require mechanical disruption with tissue homogenizers, while soft tissues (brain, liver) can be processed with gentler methods to preserve protein integrity. For formalin-fixed paraffin-embedded (FFPE) tissues, extend antigen retrieval times (up to 30 minutes) compared to cell preparations to overcome fixation-induced epitope masking . When comparing SUCLG2 expression across tissues with different mitochondrial content, normalize to mitochondrial markers (VDAC, COX IV) rather than total protein or housekeeping genes . For immunofluorescence in tissues with high autofluorescence (brain, liver), implement additional quenching steps with Sudan Black B or specialized commercial reagents. For single-cell analyses in heterogeneous tissues, combine SUCLG2 antibody staining with cell-type-specific markers to identify expression patterns in specific cell populations. Finally, when studying organ systems with known metabolic zonation (liver, kidney), use spatial analysis techniques to correlate SUCLG2 expression with metabolic gradients across tissue regions .

How can SUCLG2 antibodies facilitate the development of targeted cancer therapies?

SUCLG2 antibodies can significantly accelerate the development of targeted cancer therapies through multiple strategic applications in preclinical and translational research. Patient stratification represents a primary application—using validated SUCLG2 antibodies in tissue microarrays can identify cancer patients with SUCLG2 overexpression who might benefit from targeted therapies, as demonstrated by survival correlation studies in LUAD patients . For drug discovery, researchers can develop high-throughput screening assays employing SUCLG2 antibodies to identify compounds that modulate SUCLG2 protein levels, enzyme activity, or post-translational modifications. Mechanistic investigations using co-immunoprecipitation with SUCLG2 antibodies can reveal protein-protein interactions critical for SUCLG2 stability and function, potentially identifying additional druggable targets like the SIRT5-TRIM21 regulatory axis . For therapeutic antibody development, SUCLG2 antibodies can help characterize epitope accessibility in intact cells and tissues, identifying regions suitable for antibody-drug conjugate targeting if evidence emerges for SUCLG2 expression on cell surfaces in cancer cells. In translational research, combining SUCLG2 immunohistochemistry with metabolic imaging techniques could develop companion diagnostics that predict response to metabolic-targeted therapies. For monitoring treatment efficacy, antibodies detecting specific post-translational modifications of SUCLG2 (particularly succinylation at Lys93) could serve as pharmacodynamic biomarkers indicating successful target engagement of drugs designed to modulate this pathway . These diverse applications position SUCLG2 antibodies as versatile tools spanning the continuum from basic research to clinical translation.

What emerging technologies could enhance SUCLG2 antibody-based research in the future?

Emerging technologies promise to revolutionize SUCLG2 antibody-based research by enabling more precise, dynamic, and comprehensive analyses of this important metabolic regulator. Single-cell proteomics technologies adapted for antibody-based detection will allow researchers to examine SUCLG2 expression heterogeneity within tumors, potentially revealing subpopulations with distinct metabolic vulnerabilities . Advances in super-resolution microscopy (STORM, PALM, STED) combined with SUCLG2 antibodies will provide nanoscale visualization of SUCLG2 distribution within mitochondrial subcompartments, offering insights into its spatial regulation . For temporal studies, engineered antibody fragments labeled with photoconvertible fluorophores could enable real-time tracking of SUCLG2 dynamics in living cells. Proximity-dependent labeling approaches (BioID, APEX) using SUCLG2 as bait will map its complete protein interaction network beyond currently known partners like SIRT5 and TRIM21 . For clinical applications, multiplexed immunofluorescence platforms (Vectra, CODEX, MIBI) will allow simultaneous detection of SUCLG2 alongside dozens of other cancer and immune markers in patient samples, revealing complex associations with tumor microenvironment features. Mass cytometry (CyTOF) adapted for intracellular metabolic proteins could provide high-dimensional analysis of SUCLG2 in relation to cellular phenotypes across large patient cohorts. Finally, the integration of antibody-based detection with spatial transcriptomics and metabolomics will create multi-omic maps revealing how SUCLG2 expression patterns correlate with local metabolic activities and gene expression programs across tumor regions, offering unprecedented insights into its role in cancer metabolism .

What are the unexplored aspects of SUCLG2 biology that require future antibody-based investigations?

Despite significant advances in understanding SUCLG2 function, several critical aspects of its biology remain unexplored and warrant future antibody-based investigations. First, the tissue-specific expression patterns and functions of SUCLG2 beyond cancer contexts require systematic mapping using validated antibodies across normal human and model organism tissues . Second, the potential non-mitochondrial localization and functions of SUCLG2 remain unexamined—antibody-based subcellular fractionation studies could reveal unexpected localizations and novel functions . Third, the regulation of SUCLG2 during development and aging represents an untapped research area—immunohistochemical analyses across developmental stages and age groups could identify critical temporal expression patterns. Fourth, post-translational modifications beyond succinylation (phosphorylation, acetylation, methylation) may regulate SUCLG2 and could be investigated using modification-specific antibodies or combined antibody-mass spectrometry approaches . Fifth, the potential role of SUCLG2 in immune cell metabolism and function remains unexplored—antibody-based flow cytometry could characterize SUCLG2 expression across immune cell subtypes and activation states. Sixth, the existence and significance of SUCLG2 isoforms or splice variants has not been thoroughly investigated—isoform-specific antibodies could reveal differential expression patterns. Finally, the potential involvement of SUCLG2 in neurodegenerative diseases with mitochondrial dysfunction components represents an important research direction, as connections between cancer metabolism and neurodegeneration continue to emerge . These investigations would significantly expand our understanding of SUCLG2 biology beyond its established role in cancer metabolism.