SUCLA2 Antibody

What is SUCLA2 and what is its function in cellular metabolism?

SUCLA2 is the ATP-specific β-subunit of succinyl-CoA ligase (SCL), a crucial enzyme in the tricarboxylic acid cycle. This 463 amino acid protein is located in the mitochondrial matrix where it dimerizes with the SCS alpha subunit (SUCLG1) to form the succinyl-CoA synthetase complex. This complex catalyzes the conversion of succinyl-CoA to succinate while generating ATP in the process, representing a key step in cellular energy production. The enzyme has a calculated molecular weight of 50 kDa, though it is typically observed at 48-50 kDa in experimental conditions . Mutations in SUCLA2 are associated with mitochondrial DNA depletion syndromes (MDSs), characterized by reduced mitochondrial DNA copy numbers in affected tissues, which can lead to severe clinical manifestations including progressive external ophthalmoplegia, ataxia-neuropathy, and mitochondrial neurogastrointestinal encephalomyopathy .

What types of SUCLA2 antibodies are commercially available for research applications?

Based on the search results, there are at least two major types of SUCLA2 antibodies available for research:

• Rabbit polyclonal antibody (e.g., Proteintech 12627-1-AP): This antibody targets SUCLA2 and is applicable for Western blot, immunohistochemistry, immunofluorescence, immunoprecipitation, and ELISA applications. It shows reactivity with human, mouse, and rat samples .

• Mouse monoclonal antibody (e.g., Santa Cruz A-9): This is an IgG1 kappa light chain antibody that detects SUCLA2 protein of mouse, rat, and human origin. It is available in non-conjugated and various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates .

The choice between polyclonal and monoclonal antibodies depends on the specific experimental requirements, with polyclonals offering broader epitope recognition and monoclonals providing higher specificity for a single epitope.

What are the recommended dilutions for SUCLA2 antibody in different applications?

The optimal dilution of SUCLA2 antibody varies depending on the specific application. For the Proteintech polyclonal antibody (12627-1-AP), the following dilutions are recommended:

It is important to note that these are general recommendations, and the optimal dilution should be determined experimentally for each specific application and sample type. The manufacturer suggests that "this reagent should be titrated in each testing system to obtain optimal results" as the optimal dilution can be sample-dependent .

How should SUCLA2 antibodies be stored to maintain optimal activity?

For optimal storage of SUCLA2 antibody, the following conditions are recommended based on the Proteintech product information:

• Store the antibody at -20°C

• The antibody is stable for one year after shipment when stored properly

• Aliquoting is unnecessary for -20°C storage

• The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations (20μl sizes) may contain 0.1% BSA

Proper storage is essential to maintain antibody activity and specificity. Avoiding repeated freeze-thaw cycles is generally recommended for all antibodies, though the specific product information suggests aliquoting may not be necessary for this particular antibody.

What positive controls should be used when validating SUCLA2 antibody specificity?

When validating SUCLA2 antibody specificity, researchers should consider the following positive controls based on validated reactivity:

For Western blot applications, the following samples have been tested and validated as positive controls:

• Human cell lines: HEK-293 cells, L02 cells, SH-SY5Y cells, HepG2 cells, PC-3 cells

• Mouse tissues: brain tissue, liver tissue

For immunoprecipitation:

• HEK-293 cells have been validated

For immunohistochemistry:

• Human tissues: colon cancer tissue, heart tissue, kidney tissue

• Note that antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative

For immunofluorescence/ICC:

• HepG2 cells have been validated

Using knockout or knockdown experimental systems can provide excellent negative controls, as indicated by the multiple publications cited in the search results that used KD/KO approaches .

How can I optimize antigen retrieval for SUCLA2 immunohistochemistry?

For optimal antigen retrieval in SUCLA2 immunohistochemistry, the following protocol is recommended based on the product information:

• Primary recommended method: Use TE buffer at pH 9.0 for antigen retrieval

• Alternative method: Citrate buffer at pH 6.0 can also be effective

The choice between these methods may depend on the specific tissue type and fixation methods used. For formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval (HIER) using these buffers is typically performed.

For the immunohistochemistry procedure itself, the following steps are generally recommended:

• Deparaffinize and rehydrate tissue sections

• Perform antigen retrieval using the methods described above

• Block endogenous peroxidase (for HRP-based detection) and non-specific binding

• Incubate with SUCLA2 primary antibody at a dilution of 1:50-1:500

• Apply appropriate detection system

• Counterstain, dehydrate, and mount

The optimal protocol should be determined empirically for each experimental system and tissue type.

What are the common issues encountered when using SUCLA2 antibody in Western blot, and how can they be addressed?

Several common issues may arise when using SUCLA2 antibody in Western blot applications. Here are potential problems and their solutions:

• Multiple bands or non-specific binding:

  • Increase antibody dilution (try 1:6000 instead of 1:1000)

  • Optimize blocking conditions (try 5% non-fat dry milk or 5% BSA)

  • Increase washing duration and number of washes

  • Verify sample preparation (ensure complete denaturation)

• Weak or no signal:

  • Decrease antibody dilution (try 1:1000 instead of 1:6000)

  • Increase protein loading

  • Extend exposure time

  • Check transfer efficiency

  • Verify that your sample expresses SUCLA2 (the antibody has been validated in specific cell lines and tissues as mentioned earlier)

• Unexpected molecular weight:

  • SUCLA2 has a calculated molecular weight of 50 kDa, but is typically observed at 48-50 kDa

  • Post-translational modifications like succinylation (as mentioned in result ) may affect migration

  • Alternative splicing may generate different isoforms (result mentions two isoforms)

• Batch-to-batch variability:

  • Run appropriate positive controls with each experiment

  • Consider using monoclonal antibodies which typically show less batch-to-batch variability

For optimal Western blot results, researchers should be aware that SUCLA2 knockout/knockdown models have been used successfully in at least 3 publications, which can serve as negative controls to validate antibody specificity .

How can SUCLA2 antibodies be used to study protein succinylation in disease models?

SUCLA2 mutations have been shown to cause global protein succinylation, which contributes to the pathogenesis of mitochondrial diseases. Researchers can use SUCLA2 antibodies in conjunction with anti-succinyl-lysine antibodies to investigate this phenomenon through several approaches:

• Comparative protein succinylation profiling:

  • Western blot analysis using anti-succinyl-lysine antibodies can detect increased global protein succinylation in SUCLA2-deficient models compared to controls

  • In patient-derived cell lines, non-proliferative serum-deprived fibroblasts and differentiated myotubes showed approximately 8-fold increase in global protein succinylation compared to controls, while proliferating fibroblasts showed a 1.9-fold increase

• Identification of specific succinylated targets:

  • Immunoprecipitation with SUCLA2 antibody followed by mass spectrometry

  • SUCLA2 itself carries six succinylated lysines within its nucleotide grasp-domains, and these residues showed SIRT5-dependent changes up to 25-fold

  • Specifically, Lys108, Lys116, and Lys143 in the SUCLA2 subunit are highly conserved among species and are potential succinylation sites

• Correlation with disease progression:

  • Immunohistochemistry using SUCLA2 antibody in tissue samples from patients or disease models

  • Analysis of post-mitotic cells, which appear to accumulate more protein succinylation than proliferating cells

This research direction is particularly valuable because global protein succinylation has been identified as a biochemical hallmark in SUCLA2 patients, potentially contributing to disease pathogenesis.

What experimental approaches can be used to investigate SUCLA2 dysfunction in mitochondrial disease models?

Investigating SUCLA2 dysfunction in mitochondrial disease models requires a multifaceted approach:

• Generation of SUCLA2-deficient models:

  • Conditional knockout mouse models, such as the muscle-specific Sucla2 knockout using the Cre-Lox system with the human skeletal actin (HSA) promoter driving Cre-recombination

  • CRISPR-Cas9 gene editing to create cell lines with SUCLA2 mutations

  • Patient-derived cell lines from individuals with SUCLA2 mutations

• Validation of SUCLA2 deficiency:

  • RT-qPCR to assess transcript levels

  • Western blot with SUCLA2 antibody to confirm protein reduction

  • Enzyme activity assays using mass spectrometry to quantify functional reduction

• Phenotypic characterization:

  • Whole-body phenotyping in animal models (e.g., body weight, grip strength, spontaneous exercise)

  • Ex vivo contractility experiments on muscles (e.g., specific tetanic force, contraction and relaxation rates)

  • Histological and immunofluorescence studies to assess muscle fiber-specific phenotypes

• Mitochondrial analyses:

  • COX and SDH staining to assess mitochondrial function

  • Quantification of mitochondrial DNA copy number

  • Analysis of mitochondrial structural changes using electron microscopy

  • Measurement of oxygen consumption and ATP production

In the muscle-specific Sucla2 knockout model described in the search results, significant phenotypic changes were observed, including 95% reduction in SUCLA2 protein, 44% reduction in body weight by 3 weeks of age, 34-40% reduced grip strength, and 88% less time spent on a running wheel compared to controls. Additionally, muscle-specific effects were noted, with SOL muscles generating 40% less specific tetanic force and showing slower contraction and relaxation rates, along with a threefold increase in mitochondria .

How does SUCLA2 antibody specificity compare across species, and what are the implications for comparative studies?

SUCLA2 antibody specificity across species is an important consideration for comparative studies. Based on the search results:

• Cross-reactivity profile:

  • Both the Proteintech polyclonal antibody (12627-1-AP) and the Santa Cruz monoclonal antibody (A-9) show reactivity with human, mouse, and rat samples

  • This cross-reactivity suggests high conservation of epitopes across these species

• Evolutionary conservation implications:

  • The high conservation of key lysine residues (e.g., Lys108, Lys116, and Lys143) in SUCLA2 across species suggests functional importance

  • This conservation facilitates translational research between animal models and human studies

• Experimental design considerations:

  • When designing comparative studies, researchers should validate the antibody in each species of interest

  • Western blot may show slight variations in molecular weight between species

  • Species-specific positive controls should be included (e.g., mouse brain tissue for mouse studies, human cell lines for human studies)

• Application-specific differences:

  • For immunohistochemistry applications, species-specific differences in tissue processing and antigen retrieval may need to be optimized

  • For Western blot, the recommended dilution range (1:1000-1:6000) provides flexibility to optimize for different species

What is the significance of studying SUCLA2 in the context of mitochondrial DNA depletion syndromes (MDSs)?

Studying SUCLA2 in the context of mitochondrial DNA depletion syndromes (MDSs) is significant for several reasons:

• Genetic basis of MDSs:

  • More than 50 patients with mutations in SUCLA2 (encoding the ATP-specific β-subunit) have been reported

  • Over 20 patients with mutations in SUCLG1 (encoding the α-subunit) have been identified

  • Interestingly, no disease-causing mutations in SUCLG2 (encoding the GTP-specific β-subunit) have been described

• Molecular mechanisms:

  • SUCLA2 deficiency leads to abnormal accumulation of succinyl-CoA

  • This results in global protein succinylation, which has been identified as a biochemical hallmark in SUCLA2 patients

  • Post-mitotic cells (like neurons and differentiated myotubes) appear particularly susceptible to increased protein succinylation

• Clinical manifestations:

  • Patients with SUCLA2 mutations develop severe symptoms including progressive external ophthalmoplegia, ataxia-neuropathy, and mitochondrial neurogastrointestinal encephalomyopathy

  • These conditions are characterized by reduced mitochondrial DNA copy numbers in affected tissues

• Research models:

  • The recently developed muscle-specific Sucla2 knockout mouse model provides an in vivo system to study SCS-deficient pathogenesis in postnatal skeletal muscle

  • This model exhibits features consistent with mitochondrial myopathy, including reduced body weight, decreased grip strength, impaired muscle function, and altered mitochondrial content

• Therapeutic implications:

  • Understanding the role of SUCLA2 and protein succinylation may lead to novel therapeutic approaches

  • The identification of specific succinylation sites (e.g., Lys108, Lys116, and Lys143) provides potential targets for intervention

Research in this area has significant translational potential, as findings from animal models may inform our understanding and treatment of human mitochondrial diseases associated with SUCLA2 deficiency.

How should samples be prepared for optimal detection of SUCLA2 in different applications?

Sample preparation is critical for successful detection of SUCLA2 across different applications. Here are specific recommendations:

For Western Blot:

• Cell lysis: Use RIPA buffer or other appropriate lysis buffers containing protease inhibitors

• Tissue homogenization: For tissues like brain or liver, mechanical homogenization in appropriate buffer is recommended

• Protein denaturation: Heat samples with reducing SDS sample buffer at 95°C for 5 minutes

• Loading amount: 20-50 μg of total protein per lane is typically sufficient

• Validated samples: HEK-293 cells, L02 cells, SH-SY5Y cells, HepG2 cells, PC-3 cells, mouse brain tissue, and mouse liver tissue have all shown positive results

For Immunoprecipitation:

• Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• HEK-293 cells have been validated for successful IP with SUCLA2 antibody

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin is standard

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

• Validated tissues: Human colon cancer tissue, heart tissue, and kidney tissue have shown positive results

For Immunofluorescence:

• Fixation: 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

• Blocking: Use 0.5% BSA and 10% goat serum in 1X PBS for 1 hour

• Primary antibody dilution: 1:200-1:800

• HepG2 cells have been validated for IF applications

For analyzing patient samples:

• The culture conditions significantly affect protein succinylation levels

• Non-proliferative serum-deprived fibroblasts and differentiated myotubes show higher levels of protein succinylation (~8-fold) compared to controls

• Proliferating fibroblasts show less dramatic changes (1.9-fold increase)

These sample preparation guidelines should be adjusted based on the specific experimental requirements and further optimized for each laboratory setting.

What controls and validation steps are essential when studying SUCLA2 in knockout or knockdown models?

When studying SUCLA2 in knockout or knockdown models, the following controls and validation steps are essential:

• Validation of SUCLA2 depletion:

  • Transcript level: RT-qPCR to confirm reduction of SUCLA2 mRNA

  • Protein level: Western blot with SUCLA2 antibody to confirm protein reduction (95% reduction was observed in the muscle-specific knockout model)

  • Functional assessment: Enzyme activity assays to confirm reduced SUCLA2 function

• Appropriate controls:

  • Wild-type controls: Age-matched and sex-matched wild-type animals or cells

  • Heterozygous controls: When available, to assess gene dosage effects

  • Tissue-specific controls: In conditional knockouts, unaffected tissues serve as internal controls

  • Rescue experiments: Re-expression of SUCLA2 to confirm phenotype reversal

• Phenotypic characterization:

  • Comprehensive phenotyping: In animal models, measure parameters like body weight, grip strength, and exercise capacity

  • Tissue-specific analyses: For muscle-specific knockouts, assess contractile function (specific tetanic force, contraction/relaxation rates)

  • Histological analysis: Assess tissue morphology, fiber type composition, and mitochondrial content

  • Molecular profiling: Analyze protein succinylation patterns, which are expected to increase in SUCLA2-deficient models

• Functional assessments:

  • Mitochondrial function: COX and SDH staining, oxygen consumption measurements

  • Energy metabolism: ATP production, metabolite analysis

  • Stress responses: Assessment of cellular responses to metabolic challenges

• Temporal considerations:

  • Developmental effects: In constitutive knockouts, assess effects throughout development

  • Age-dependent changes: In conditional models, monitor changes over time after gene inactivation

The muscle-specific Sucla2 knockout described in the search results employed many of these validation approaches, confirming 95% reduction in SUCLA2 protein and demonstrating significant phenotypic effects including reduced body size, grip strength, and altered muscle function . Such comprehensive validation ensures that observed phenotypes can be confidently attributed to SUCLA2 deficiency.

How can SUCLA2 antibodies be used to investigate protein-protein interactions within the succinyl-CoA synthetase complex?

SUCLA2 antibodies can be valuable tools for investigating protein-protein interactions within the succinyl-CoA synthetase complex through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use SUCLA2 antibody to immunoprecipitate the protein along with its binding partners

  • Western blot analysis of the immunoprecipitated material can identify interacting proteins such as SUCLG1 (the α-subunit of the complex)

  • The search results indicate that SUCLA2 antibody has been validated for IP applications in HEK-293 cells

• Proximity ligation assay (PLA):

  • This technique allows visualization of protein-protein interactions in situ

  • Requires two primary antibodies (e.g., anti-SUCLA2 and anti-SUCLG1) from different species

  • The validated reactivity of SUCLA2 antibodies with human, mouse, and rat samples facilitates cross-species studies

• Immunofluorescence co-localization:

  • SUCLA2 antibody can be used at 1:200-1:800 dilution for immunofluorescence applications

  • Co-staining with antibodies against other complex components or mitochondrial markers

  • HepG2 cells have been validated for IF applications with SUCLA2 antibody

• Analysis of succinylation sites:

  • The search results indicate that SUCLA2 carries six succinylated lysines within its nucleotide grasp-domains

  • Lys108, Lys116, and Lys143 in the SUCLA2 subunit are highly conserved among species

  • These lysines can be mapped to the protein structure of SCL to understand their functional significance

• Cross-linking studies:

  • Chemical cross-linking combined with immunoprecipitation using SUCLA2 antibody

  • Mass spectrometry analysis of cross-linked peptides to identify interaction interfaces

These methods, used individually or in combination, can provide insights into how SUCLA2 interacts with other components of the succinyl-CoA synthetase complex and how these interactions may be affected in disease states. The high conservation of key lysine residues suggests their potential importance in maintaining protein structure and function across species.

What are the considerations for using SUCLA2 antibody in metabolic flux analysis and mitochondrial function studies?

When using SUCLA2 antibody in metabolic flux analysis and mitochondrial function studies, researchers should consider several important factors:

• Relationship between SUCLA2 levels and TCA cycle function:

  • SUCLA2 is a critical component of the succinyl-CoA synthetase complex in the TCA cycle

  • Quantification of SUCLA2 protein levels (via Western blot) can be correlated with metabolic flux measurements

  • In Sucla2 knockout models, significant changes in contractile function and metabolic parameters have been observed

• Integration with other metabolic markers:

  • Combined immunostaining for SUCLA2 and other mitochondrial proteins

  • In the muscle-specific Sucla2 knockout model, researchers observed:

    • Threefold increase in mitochondrial content in SOL muscles

    • Increased staining for both COX and SDH (mitochondrial enzymes)

    • Nearly doubled proportion of Type 1 myosin heavy chain expressing fibers

• Tissue-specific considerations:

  • Different tissues show varying dependency on SUCLA2 function

  • The muscle-specific knockout showed more pronounced effects in slow-twitch (SOL) muscles compared to fast-twitch (EDL) muscles

  • When designing experiments, consider tissue-specific expression patterns and metabolic requirements

• Correlation with protein succinylation:

  • SUCLA2 deficiency leads to increased global protein succinylation

  • This can affect the function of multiple metabolic enzymes

  • Consider using anti-succinyl-lysine antibodies alongside SUCLA2 antibody to correlate SUCLA2 levels with protein succinylation status

• Technical considerations for mitochondrial studies:

  • Sample preparation is critical for maintaining mitochondrial integrity

  • For immunofluorescence studies of mitochondrial proteins, ensure proper permeabilization

  • When examining SUCLA2 in isolated mitochondria, consider subfractionation approaches to distinguish matrix proteins

• Metabolic flux analysis integration:

  • Stable isotope tracing (e.g., 13C-labeled substrates) can be combined with SUCLA2 immunoprecipitation

  • This approach can help determine how SUCLA2 levels or mutations affect specific metabolic pathways

  • Consider the impact of post-translational modifications (such as succinylation) on enzyme activity

These considerations highlight the importance of integrating SUCLA2 antibody-based analyses with functional metabolic studies to gain comprehensive insights into mitochondrial function in both normal and disease states.

How might advances in SUCLA2 antibody technology contribute to diagnostic applications for mitochondrial diseases?

Advances in SUCLA2 antibody technology could significantly contribute to diagnostic applications for mitochondrial diseases in several ways:

• Development of diagnostic immunoassays:

  • Highly specific SUCLA2 antibodies could be used to develop ELISA or other immunoassays to detect abnormal SUCLA2 levels in patient samples

  • Such assays might serve as screening tools for mitochondrial disorders associated with SUCLA2 dysfunction

  • Both polyclonal and monoclonal antibodies have shown utility in ELISA applications

• Protein succinylation as a biomarker:

  • Research has shown that SUCLA2 mutations cause global protein succinylation, which could serve as a diagnostic marker

  • Antibodies that detect SUCLA2 and succinylated proteins could be used in combination to assess this biochemical hallmark

  • Non-proliferative cells (like differentiated myotubes) showed approximately 8-fold increase in global protein succinylation in SUCLA2-deficient patient samples, suggesting potential diagnostic utility

• Immunohistochemical diagnosis:

  • SUCLA2 antibodies can be used at 1:50-1:500 dilution for immunohistochemistry

  • Tissue biopsies from patients could be analyzed for SUCLA2 expression patterns and associated changes

  • Positive IHC has been detected in human colon cancer tissue, heart tissue, and kidney tissue

• Integration with genetic testing:

  • SUCLA2 antibody-based assays could complement genetic testing to assess functional consequences of variants of uncertain significance

  • The combination of genetic data with protein expression and function analysis could improve diagnostic accuracy

• Patient stratification:

  • Different mutations in SUCLA2 may have varying effects on protein expression, stability, or function

  • Antibody-based approaches could help stratify patients based on the molecular consequences of their specific mutations

  • This could potentially guide personalized treatment approaches

The development of more specific, sensitive, and diverse SUCLA2 antibodies, including those that recognize specific post-translational modifications or conformational states, could enhance our ability to diagnose and characterize mitochondrial diseases associated with SUCLA2 dysfunction.

What research questions remain unexplored regarding the role of SUCLA2 in disease pathogenesis?

Despite significant advances in understanding SUCLA2's role in mitochondrial function and disease, several important research questions remain unexplored:

• Tissue-specific vulnerability:

  • Why do certain tissues show greater vulnerability to SUCLA2 deficiency?

  • The muscle-specific knockout model revealed more pronounced effects in slow-twitch (SOL) muscles compared to fast-twitch (EDL) muscles

  • What molecular mechanisms underlie these tissue-specific differences?

• Protein succinylation mechanisms and consequences:

  • While SUCLA2 mutations are known to cause global protein succinylation, the complete catalog of succinylated proteins and their functional alterations remains incompletely characterized

  • How do changes in specific succinylated proteins contribute to disease phenotypes?

  • Are there compensatory mechanisms to mitigate excessive protein succinylation?

• Relationship with mitochondrial DNA maintenance:

  • SUCLA2 mutations are associated with mitochondrial DNA depletion syndromes, but the mechanistic link between SUCLA2 function and mtDNA maintenance is not fully elucidated

  • What molecular pathways connect TCA cycle dysfunction with mtDNA stability?

• Therapeutic targets:

  • Can the reversal of protein succinylation (e.g., through modulation of desuccinylases like SIRT5) ameliorate disease phenotypes?

  • Are there bypass mechanisms that could compensate for SUCLA2 deficiency?

  • Could metabolic interventions restore energy balance in affected tissues?

• Developmental aspects:

  • How does SUCLA2 deficiency affect developmental processes?

  • Are there critical periods during which SUCLA2 function is particularly important?

  • The knockout mouse model showed 44% reduced body weight by just 3 weeks of age, suggesting early developmental effects

• Interaction with environmental factors:

  • How do environmental stressors, nutrition, or exercise modify the phenotypes associated with SUCLA2 dysfunction?

  • Could lifestyle interventions mitigate disease progression?

• SUCLA2 isoforms and regulation:

  • The search results mention that two isoforms of SUCLA2 arise from alternative splicing

  • How are these isoforms differentially regulated and what are their specific functions?

Addressing these research questions will require a combination of approaches, including advanced antibody-based techniques, animal models, patient-derived cells, and integrated omics analyses. The continued development and characterization of SUCLA2 antibodies will be essential for many of these investigations.