Sirt3 Antibody

What are the key characteristics of SIRT3 and why is it significant in mitochondrial research?

SIRT3 is a NAD+-dependent protein deacetylase primarily localized in mitochondria, though also found in the nucleus and cytoplasm of cardiomyocytes. It exists in two forms in humans: a full-length ~44 kDa protein and a processed ~28-30 kDa mitochondrial form that results from cleavage between Arg99-Arg100. SIRT3 is the only sirtuin whose increased expression has been correlated with extended lifespan in humans . It plays crucial roles in protecting cells against genotoxic and oxidative stress by deacetylating targets like Ku70, SOD2, and cyclophilin D . In mitochondria, SIRT3 regulates the acetylation of metabolic enzymes including acetyl-CoA synthetase 2, thereby controlling mitochondrial metabolism and function .

Which forms of SIRT3 can most commercial antibodies detect, and how should I select the appropriate antibody?

Most commercial antibodies detect the processed mitochondrial form of SIRT3 (~28-30 kDa), though some can recognize both the full-length (44 kDa) and processed forms. When selecting an antibody, consider:

For research focusing on mitochondrial functions, choose antibodies validated specifically for the 28-30 kDa form. Several antibodies from manufacturers like R&D Systems, Cell Signaling, and Proteintech have been validated for detecting the mitochondrial form in Western blot applications .

Which tissue samples are most suitable as positive controls for SIRT3 antibody validation?

Based on experimental evidence, these tissues and cell lines consistently show strong SIRT3 expression:

Including these positive controls when validating a new antibody provides reliable benchmarks for specificity and sensitivity .

How should I design experiments to investigate SIRT3's role in cellular stress response?

When studying SIRT3's role in cellular stress response, implement a multi-layered experimental approach:

• Stress induction protocols:

  • Oxidative stress: H₂O₂ treatment (concentration ranges from low/adaptive to high/cytotoxic)

  • Metabolic stress: 3-NPA treatment (model relevant to Huntington's disease)

  • Excitotoxic stress: Glutamate receptor agonists (NMDA, KA) at varying concentrations

• Comparative analysis:

  • SIRT3 wild-type versus knockout/knockdown models

  • Monitor stress responses before and after SIRT3 manipulation

  • Include time-course experiments to capture adaptive responses

• Key parameters to measure:

  • SIRT3 expression and localization changes under stress

  • ROS production and oxidative damage markers

  • Mitochondrial function (membrane potential, ATP production)

  • Acetylation status of SIRT3 targets (SOD2, cyclophilin D)

  • Cell survival and apoptotic markers

Research has demonstrated that SIRT3 levels increase in response to low/moderate stress but decrease under severe stress conditions, suggesting a biphasic response pattern .

What are effective approaches for studying SIRT3 substrate interactions and deacetylation activity?

To effectively study SIRT3 substrate interactions and deacetylation activities, employ these complementary techniques:

• For substrate identification and validation:

  • Immunoprecipitate mitochondrial proteins with anti-acetyl-lysine antibodies followed by mass spectrometry to identify acetylated proteins

  • Perform comparative acetylome analysis between wild-type and SIRT3-knockout samples

  • Use SPOT peptide libraries to determine binding preferences and substrate specificity

• For deacetylation activity assessment:

  • Monitor changes in mitochondrial protein acetylation patterns using anti-acetyl-lysine antibodies in western blots

  • Perform in vitro deacetylation assays with immunoprecipitated candidate substrates

  • Analyze functional consequences of deacetylation (e.g., enzyme activity measurements of known targets)

• For protein-protein interaction studies:

  • Co-immunoprecipitation with validated SIRT3 antibodies

  • Proximity ligation assays for in situ detection

  • Yeast two-hybrid screening for novel interactions

Research has identified several important SIRT3 targets including SOD2, IDH2, LCAD, OTC, HMGCS2, and Ku70, with deacetylation typically resulting in enhanced activity of these enzymes .

What considerations are crucial when designing knockout or knockdown studies of SIRT3?

When designing SIRT3 knockout or knockdown studies, address these critical factors:

• Model selection considerations:

  • Global knockout mice exhibit clear phenotypes in stress response but may develop compensatory mechanisms

  • Conditional tissue-specific knockouts allow investigation of tissue-dependent functions

  • Inducible systems enable temporal control to distinguish developmental from acute effects

  • Cell-specific knockdown using siRNA/shRNA provides acute effects with fewer compensatory changes

• Validation requirements:

  • Confirm knockout/knockdown efficiency at both mRNA and protein levels

  • Verify functional depletion by assessing mitochondrial protein acetylation patterns

  • Include rescue experiments with wild-type SIRT3 re-expression

• Phenotypic assessment:

  • Examine both basal and stressed conditions - SIRT3-/- mice show increased vulnerability to oxidative and excitatory stress

  • Compare multiple physiological parameters - SIRT3 knockout mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle

  • Assess tissue-specific effects - SIRT3-/- mice show increased vulnerability of striatal and hippocampal neurons in models relevant to Huntington's disease and epilepsy

Research has demonstrated that SIRT3 knockout mice exhibit hyperacetylation of mitochondrial proteins and activation of stress response pathways even under basal conditions .

Why might I detect multiple bands when using SIRT3 antibodies in Western blot?

Multiple bands in SIRT3 Western blots can result from several biological and technical factors:

To address these issues, include appropriate controls (SIRT3 knockout tissue, recombinant SIRT3 protein) and validate results with multiple antibodies targeting different epitopes .

What are the optimal sample preparation conditions for SIRT3 detection in different applications?

Optimal sample preparation conditions vary by application type:

• For Western blotting:

  • Use reducing conditions as specified in multiple protocols

  • For mitochondrial SIRT3, perform subcellular fractionation first

  • Process samples immediately on ice with protease inhibitors

  • Use Immunoblot Buffer Group 1 for consistent results

• For immunohistochemistry:

  • Recommended antigen retrieval: TE buffer pH 9.0

  • Alternative: citrate buffer pH 6.0

  • Antibody dilutions typically range from 1:50-1:800 depending on the antibody

• For immunofluorescence:

  • Fixation with 4% paraformaldehyde

  • Permeabilization with 0.1-0.5% Triton X-100

  • Co-staining with mitochondrial markers recommended to confirm localization

  • Dilutions typically in 1:50-1:200 range

• For storage conditions:

  • Most antibodies stable at -20°C for 12 months

  • Avoid repeated freeze-thaw cycles

  • For reconstituted antibodies: 6 months at -20 to -70°C

Research shows that proper sample preparation significantly impacts detection sensitivity, particularly for the mitochondrial form of SIRT3 .

How can I improve specificity when detecting SIRT3 in mitochondrial fractions?

To enhance specificity when detecting SIRT3 in mitochondrial fractions:

• Mitochondrial isolation optimization:

  • Use differential centrifugation with Percoll gradient for high purity

  • Verify fraction purity using mitochondrial markers (VDAC, COX IV) and markers for other organelles (histone H3, calnexin) to confirm minimal contamination

  • Maintain sample integrity by working quickly at 4°C with protease inhibitors

• Antibody selection strategies:

  • Choose antibodies specifically validated for mitochondrial SIRT3 detection

  • Prefer antibodies targeting epitopes in the C-terminal region (present in both forms but enriched in mitochondria)

  • Use antibodies that consistently detect the 28-30 kDa band in mitochondrial fractions

• Controls and validation:

  • Include SIRT3 knockout/knockdown samples as negative controls

  • Use tissues with high SIRT3 expression (heart, liver) as positive controls

  • Verify proper protein loading with mitochondrial housekeeping proteins

• Technical considerations:

  • Optimize antibody concentration - titrate from 1:1000 to 1:5000 for Western blots

  • Increase washing stringency to reduce non-specific binding

  • Use fresh samples where possible to minimize degradation

Research indicates that the processed 28-30 kDa form is predominant in mitochondrial fractions, while the full-length 44 kDa form may appear in nuclear fractions .

How can I effectively study changes in the acetylation status of SIRT3 target proteins?

To effectively monitor acetylation changes in SIRT3 target proteins:

• Global acetylome analysis:

  • Perform immunoprecipitation with anti-acetyl-lysine antibodies followed by mass spectrometry

  • Compare acetylation patterns between wild-type and SIRT3-/- samples

  • Use stable isotope labeling (SILAC) for quantitative comparison

• Targeted analysis of specific substrates:

  • Immunoprecipitate known SIRT3 targets (SOD2, IDH2, LCAD)

  • Probe with anti-acetyl-lysine antibodies

  • Correlate acetylation status with functional activity measurements

• Validated experimental approaches:

  • Western blot analysis using anti-acetyl-lysine antibodies shows multiple acetylated mitochondrial proteins with prominent bands at 96, 73, and 56 kDa

  • Several mitochondrial proteins show increased acetylation in skeletal muscle mitochondria from SIRT3 KO mice

  • Analysis of total mitochondrial protein acetylation demonstrates approximately 2.5-fold higher acetylation levels in SIRT3-/- hippocampal tissue compared to wild-type

• Activity-based assays:

  • Measure enzymatic activity of known SIRT3 targets

  • Correlate activity with acetylation status

  • Use site-specific acetyl-lysine antibodies when available

To distinguish SIRT3-specific effects, include proper controls and validation steps using SIRT3 knockout models or pharmacological inhibition .

What approaches can I use to investigate SIRT3's role in different subcellular compartments?

To investigate SIRT3's role in different subcellular compartments:

• Subcellular fractionation techniques:

  • Differential centrifugation to separate mitochondria, nuclei, and cytosol

  • Density gradient separation for high-purity fractions

  • Verify compartment purity with specific markers for each fraction

• Localization studies:

  • Immunofluorescence microscopy with co-localization markers

  • Super-resolution microscopy for detailed subcellular distribution

  • Live-cell imaging with fluorescently tagged SIRT3 to track dynamic changes

• Compartment-specific functional analysis:

  • For mitochondrial functions: measure oxygen consumption, ATP production, ROS generation

  • For nuclear functions: assess histone deacetylation, gene expression changes

  • For cytoplasmic role: examine interaction with cytosolic binding partners

Research shows that while the short form (28 kDa) of SIRT3 is localized exclusively in mitochondria, the long form (44 kDa) can be found in the mitochondria, nucleus, and cytoplasm of cardiomyocytes . During stress conditions, SIRT3 levels increase not only in mitochondria but also in the nuclei of cardiomyocytes .

How can I design experiments to study the regulation of SIRT3 expression under different physiological conditions?

To study regulation of SIRT3 expression under different physiological conditions:

• Physiological stimuli with documented effects on SIRT3:

  • Exercise: Running wheel exercise increases SIRT3 expression in hippocampal neurons through glutamatergic neurotransmission

  • Temperature changes: Cold exposure increases SIRT3 expression in brown adipocytes, while elevated temperatures reduce expression

  • Caloric restriction/fasting: Alters SIRT3 expression in skeletal muscle

  • Excitatory neurotransmission: Low concentrations of glutamate, NMDA, or KA elevate SIRT3 levels, while higher concentrations reduce levels

• Experimental design recommendations:

  • Implement time-course studies to capture dynamic changes

  • Include tissue-specific analyses as responses may differ between tissues

  • Compare mRNA and protein levels to distinguish transcriptional from post-transcriptional regulation

  • Use reporter gene assays with the SIRT3 promoter to identify regulatory elements

• Molecular mechanisms assessment:

  • Analyze transcription factor binding to the SIRT3 promoter using ChIP

  • Examine epigenetic modifications at the SIRT3 locus

  • Investigate post-transcriptional regulation by miRNAs

Research has demonstrated that SIRT3 is a stress-responsive protein whose expression increases under moderate stress conditions but may decrease under severe stress .

How can I investigate the relationship between SIRT3 and cellular redox homeostasis?

To investigate SIRT3's role in redox homeostasis:

• Assessment of ROS production and management:

  • Measure ROS using fluorescent indicators (DCF-DA, MitoSOX)

  • Compare ROS levels between wild-type and SIRT3-deficient models

  • Analyze expression and activity of antioxidant enzymes

• Key experimental findings:

  • SIRT3 knockout mice develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling

  • SIRT3 knockdown cells exhibit reduced mitochondrial oxidation and increased ROS production

  • SIRT3 knockdown cells show upregulation of stress response genes and enhanced activities of ROS clearance enzymes (SOD and catalase)

  • SIRT3 deacetylates SOD2, a major mitochondrial antioxidant enzyme

• Experimental approaches:

  • Challenge cells with oxidative stressors (H₂O₂, paraquat)

  • Measure oxidative damage markers (protein carbonylation, lipid peroxidation)

  • Assess mitochondrial function parameters alongside redox status

  • Perform rescue experiments with antioxidants or SOD2 overexpression

These approaches can help elucidate the molecular mechanisms by which SIRT3 regulates mitochondrial redox balance and protects against oxidative stress-induced damage .

What methodologies are appropriate for studying SIRT3's role in metabolic diseases?

For investigating SIRT3's role in metabolic diseases:

• Model systems:

  • Diet-induced obesity models

  • Genetic models of diabetes (type 1 and 2)

  • Tissue-specific SIRT3 knockout models

  • Cell culture models with metabolic challenges

• Key metabolic parameters to assess:

  • Glucose tolerance and insulin sensitivity

  • Mitochondrial respiration and ATP production

  • Fatty acid oxidation and lipid metabolism

  • Expression and acetylation status of metabolic enzymes

• Molecular pathways to investigate:

  • SIRT3-mediated deacetylation of metabolic enzymes

  • JNK activation status and insulin signaling components

  • IRS-1 phosphorylation patterns (serine vs. tyrosine)

  • Mitochondrial dynamics and quality control mechanisms

Research has shown that SIRT3 expression in skeletal muscle is decreased in models of both type 1 and type 2 diabetes . SIRT3 knockout mice exhibit decreased oxygen consumption, and SIRT3 knockdown in cultured myoblasts results in reduced mitochondrial oxidation, increased ROS, activation of JNK, altered IRS-1 phosphorylation, and decreased insulin signaling .

How can I design studies to investigate SIRT3's contribution to aging and longevity?

To investigate SIRT3's contribution to aging and longevity:

• Experimental models:

  • Longitudinal studies with SIRT3 knockout/transgenic mice

  • Cell senescence models (replicative and stress-induced)

  • Tissue samples from young versus aged subjects

  • Human genetic association studies with longevity cohorts

• Age-related parameters to measure:

  • Lifespan and healthspan metrics

  • Mitochondrial function across age

  • ROS production and oxidative damage accumulation

  • Expression and activity of SIRT3 with aging

  • Acetylation status of key SIRT3 targets during aging

• Intervention studies:

  • Caloric restriction effects on SIRT3 expression and function

  • Exercise interventions to modulate SIRT3 activity

  • NAD+ precursor supplementation

SIRT3 is particularly relevant to aging research as it is the only sirtuin whose increased expression has been shown to correlate with extended lifespan in humans . Additionally, SIRT3's protective effects against oxidative stress and its role in maintaining mitochondrial function are highly relevant to theories of aging that emphasize mitochondrial decline and oxidative damage accumulation .