Acetyl-SOD2 (K68) Recombinant Monoclonal Antibody

What is the biological significance of SOD2 K68 acetylation?

SOD2 (manganese superoxide dismutase) is a critical mitochondrial antioxidant enzyme responsible for converting superoxide radicals into hydrogen peroxide and molecular oxygen. Acetylation at lysine 68 (K68) serves as a post-translational regulatory mechanism that significantly decreases SOD2's dismutase activity. This acetylation directly affects the enzyme's ability to coordinate with Mn³⁺, which is essential for its catalytic function. Studies have demonstrated that SOD2 purified from cells treated with deacetylase inhibitors (TSA and NAM) shows approximately 50% reduction in specific activity compared to control samples, correlating with increased acetylation levels . This post-translational modification represents a rapid responsive mechanism for cells to regulate mitochondrial redox status without altering gene expression.

How does SIRT3 regulate SOD2 activity through K68 deacetylation?

SIRT3, a NAD⁺-dependent mitochondrial deacetylase, directly interacts with, deacetylates, and activates SOD2. In response to oxidative stress, SIRT3 transcription increases, leading to enhanced SOD2 deacetylation and subsequent activation . This relationship creates a responsive feedback mechanism: rising reactive oxygen species (ROS) levels trigger SIRT3 upregulation, which then deacetylates SOD2 at K68, restoring its antioxidant capacity. Experimental evidence shows that SOD2-mediated ROS reduction is synergistically enhanced by SIRT3 co-expression but is negated when SIRT3 is depleted . This SIRT3-SOD2 axis represents a crucial adaptive response to oxidative stress in mitochondria.

What are the primary applications for Acetyl-SOD2 (K68) Recombinant Monoclonal Antibody?

This specialized antibody is primarily used for detecting human SOD2 protein acetylated specifically at K68 in research applications. According to validated protocols, its applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of acetylated SOD2 in biological samples.

• Immunohistochemistry (IHC): For visualization of acetylated SOD2 in tissue sections, with a recommended dilution of 1:50-1:200 .

The antibody has been specifically validated for human samples and demonstrated successful IHC application in human breast cancer tissue, as evidenced by images showing specific staining when used with the Leica BondTM system .

How can the Acetyl-SOD2 (K68) antibody be used to investigate the role of oxidative stress in vascular pathologies?

The antibody serves as a critical tool for investigating vascular oxidative stress mechanisms. Research has established that Sirt3 expression declines by approximately 40% by age 65, correlating with increased hypertension incidence . This decline leads to SOD2 hyperacetylation at K68, which has been directly linked to endothelial dysfunction and hypertension development.

For vascular research applications, the antibody can be utilized to:

• Quantify SOD2 K68 acetylation levels in vascular tissues from experimental models of hypertension

• Correlate acetylation status with direct measurements of mitochondrial ROS production using mitoSOX probes

• Evaluate the efficacy of interventions targeting SIRT3-SOD2 pathway

What insights can the antibody provide regarding acetylated SOD2's role in cancer stemness and metastasis?

The antibody represents a valuable tool for investigating the paradoxical role of SOD2 in cancer. While SOD2 has been established as a suppressor of tumor initiation, late-stage solid tumors often display elevated SOD2 expression . Research has revealed that overexpression of SOD2 leads to its accumulation in an acetylated form that increases mitochondrial reactive oxygen species and activates HIF2α, a potent promoter of cancer stem cell formation .

Researchers can apply this antibody to:

• Compare acetylated SOD2 levels between primary tumors and metastatic lesions

• Evaluate the correlation between SOD2 K68 acetylation and cancer stemness markers

• Monitor changes in acetylation status during therapeutic interventions

Studies using this approach have demonstrated increased SOD2 and HIF2α in metastatic lesions compared to matched primary tumors from the same patients . Additionally, recent research indicates that acetylated SOD2 may not only lose dismutase activity but gain peroxidase activity that could amplify oxidative damage to mitochondria, contributing to therapy resistance .

How can the antibody be used to explore the dual role of SOD2 acetylation in cellular protection versus pathology?

The antibody enables investigation of the "Jekyll and Hyde" nature of SOD2 acetylation. Research suggests that while SOD2 typically serves protective functions, its acetylated form at K68 can promote pathological processes . This dual nature makes the antibody valuable for studying how SOD2 function switches between protective and harmful roles in various contexts.

Experimental designs could include:

• Time-course studies examining SOD2 acetylation status during stress response and recovery phases

• Comparison of acetylation levels across different tissue types with varying metabolic demands

• Correlation of acetylation status with cellular outcomes (survival, adaptation, or death)

Research has shown that SOD2 K68 acetylation can link variations in SOD2 activity to changes in cellular phenotype. For example, down-regulation of Sirt3 increases SOD2 acetylation and molecular signatures indicative of cancer stemness, while this effect is attenuated with concomitant SOD2 knockdown . Similarly, expression of acetylation mimic (SOD2K68Q) or resistant mutant (SOD2K68R) alters cancer stem cell phenotypes in a manner consistent with SOD2 acetylation driving HIF2α expression .

What are the optimal conditions for using the Acetyl-SOD2 (K68) antibody in immunohistochemistry applications?

For optimal immunohistochemistry results, researchers should follow these validated protocols:

• Sample Preparation:

  • Paraffin embedding of tissues followed by sectioning

  • Dewaxing and hydration of sections

  • Antigen retrieval performed under high pressure in citrate buffer (pH 6.0)

  • Blocking with 10% normal goat serum for 30 minutes at room temperature

• Primary Antibody Application:

  • Dilute antibody at 1:50-1:200 in solution containing 1% BSA

  • Incubate overnight at 4°C

  • Store antibody at -20°C or -80°C when not in use, avoiding repeated freeze-thaw cycles

• Detection System:

  • Use of goat anti-rabbit polymer IgG labeled by HRP

  • Visualization with 0.69% DAB

  • Counterstaining as appropriate for the target tissue

These conditions have been successfully applied for detecting acetylated SOD2 in human breast cancer tissues, with clear specific staining observed when following this protocol .

What experimental controls should be included when using this antibody to study SOD2 acetylation?

To ensure reliable and interpretable results, the following controls should be incorporated:

• Positive Controls:

  • Samples treated with deacetylase inhibitors (TSA and NAM) to increase acetylation levels

  • Cell lines or tissues with confirmed high SIRT3 expression for negative acetylation control

  • Human breast cancer tissue sections, which have been validated for positive staining

• Negative Controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at matching concentration)

  • Non-human tissues (as the antibody is specific to human SOD2)

• Specificity Controls:

  • Comparison with total SOD2 antibody staining

  • Pretreatment of samples with CobB (bacterial deacetylase) to reduce acetylation

  • Expression of SOD2 K68R (acetylation-resistant) and SOD2 K68Q (acetylation-mimetic) mutants

The specificity of acetyl-K68-SOD2 antibodies has been previously validated in mouse models with reduced Sirt3 expression and through site-directed mutagenesis of K68 in cell systems .

What methods can be used to quantify changes in SOD2 activity in relation to K68 acetylation status?

Several complementary approaches can be employed to correlate SOD2 acetylation with its enzymatic activity:

• Direct Enzyme Activity Assay:

  • Immunopurification of SOD2 from experimental samples

  • Measurement of specific dismutase activity using established biochemical assays

  • Correlation with acetylation status determined by Western blotting with the Acetyl-SOD2 (K68) antibody

• Comparative Analysis of Site-Specific Mutants:

  • Expression of wild-type SOD2, SOD2 K68Q (acetylation mimetic), and SOD2 K68R (acetylation resistant)

  • Measurement of enzyme activity and functional outcomes

  • Analysis of mitochondrial ROS levels using probes such as mitoSOX

• Manipulating Acetylation Status:

  • Treatment with deacetylase inhibitors (TSA and NAM) to increase acetylation

  • Expression or knockdown of SIRT3 to modulate deacetylation

  • In vitro deacetylation using purified CobB and NAD+

Previous studies have demonstrated that SOD2 purified from cells treated with deacetylase inhibitors showed approximately 50% decrease in specific activity compared to control samples. Similarly, the acetylation mimetic mutant SOD2 K68Q exhibited about 60% decrease in activity compared to wild-type SOD2 .

How does SOD2 K68 acetylation contribute to hypertension and vascular dysfunction?

SOD2 K68 acetylation plays a central role in vascular oxidative stress that contributes to hypertension pathophysiology through multiple mechanisms:

The pathway connecting SOD2 acetylation to hypertension involves:

• Reduced SOD2 activity due to K68 acetylation

• Increased mitochondrial superoxide levels

• Endothelial dysfunction

• Vascular remodeling

• Sustained blood pressure elevation

This mechanistic understanding suggests therapeutic approaches targeting SIRT3 activity or directly modulating SOD2 acetylation could provide novel treatments for hypertension.

What is the evidence for SOD2 K68 acetylation's role in cancer progression and treatment resistance?

SOD2 K68 acetylation has emerged as a critical factor in cancer biology, particularly regarding cancer stem cell formation and treatment resistance:

• Stem Cell Reprogramming:
  Overexpression of SOD2 causes its accumulation in an acetylated form that increases mitochondrial ROS and activates HIF2α, a potent promoter of cancer stem cell formation . This process contributes to treatment failure and metastatic recurrence in breast cancer.

• Clinical Correlation:
  Increased SOD2 and HIF2α expression has been documented in metastatic lesions compared with primary tumors from the same patients, supporting the role of acetylated SOD2 in cancer progression .

• Functional Transformation:
  Recent studies indicate that acetylated SOD2 not only loses dismutase activity but gains peroxidase activity that amplifies oxidative damage to mitochondria and increases ROS generation . This functional switch represents a remarkable example of how post-translational modifications can fundamentally alter enzyme function.

• Treatment Resistance Mechanism:
  Acetylated SOD2 at K68 endows breast cancer cells with resistance to tamoxifen by increasing H₂O₂ levels, a well-established characteristic of cancer stem cells .

• Down-regulation of Sirt3 increasing SOD2 acetylation and cancer stemness markers

• Silencing of mitochondrial acetyl transferase GCN5L1 decreasing acetylation and reducing cancer stem cell markers

• Expression of SOD2 K68Q (acetylation mimic) and SOD2 K68R (acetylation resistant) mutants altering cancer stem cell phenotypes

How can SOD2 K68 acetylation status be used as a biomarker for disease progression or treatment response?

SOD2 K68 acetylation status holds significant potential as a biomarker across multiple disease contexts:

• Cardiovascular Disease:

  • Elevated SOD2 K68 acetylation in vascular tissues correlates with endothelial dysfunction and hypertension development

  • Longitudinal monitoring could predict hypertension risk in aging populations

  • Response to antihypertensive therapies may correlate with normalization of acetylation levels

• Cancer Progression:

  • Increased SOD2 K68 acetylation in primary tumors may predict metastatic potential

  • Comparison between primary and metastatic lesions shows elevated acetylated SOD2 in metastases

  • Changes in acetylation status could serve as an early indicator of treatment response

• Metabolic Disorders:

  • SOD2 hyperacetylation correlates with metabolic conditions that increase NADH and acetyl-CoA levels

  • Monitoring SOD2 acetylation could help assess mitochondrial health in metabolic syndrome

Implementation for biomarker development requires:

• Standardized protocols for sample collection and processing

• Quantitative assays using the Acetyl-SOD2 (K68) antibody

• Correlation with clinical outcomes in longitudinal studies

• Integration with other established biomarkers

The specificity of the Acetyl-SOD2 (K68) Recombinant Monoclonal Antibody makes it particularly valuable for translational research aiming to develop such biomarkers, as it provides precise detection of this specific post-translational modification .

What experimental approaches can distinguish between the effects of SOD2 expression levels versus acetylation status?

Distinguishing between the effects of SOD2 expression versus its acetylation status requires carefully designed experiments that can separate these variables:

• Expression of Site-Specific Mutants:

  • Wild-type SOD2: Natural regulation of acetylation

  • SOD2 K68R: Expression with blocked acetylation (acetylation-resistant)

  • SOD2 K68Q: Expression with constitutive acetylation mimic

  These constructs allow researchers to maintain consistent expression levels while isolating the effects of acetylation status. Studies have shown that SOD2 K68Q exhibits approximately 60% decrease in specific activity compared to wild-type SOD2, while SOD2 K68R shows minimal effects on enzyme activity .

• Modulation of Acetyltransferase/Deacetylase Activity:

  • SIRT3 silencing or overexpression

  • GCN5L1 (mitochondrial acetyltransferase) manipulation

  • Treatment with deacetylase inhibitors (TSA and NAM)

  Analysis should include quantification of both total SOD2 and acetylated SOD2 levels to account for potential feedback effects on expression. Notably, SIRT3 silencing has been shown to both increase acetylation and alter SOD2 expression, suggesting coordinated regulation .

• In Vitro Deacetylation:

  • Immunopurification of SOD2 from experimental samples

  • Treatment with bacterial deacetylase CobB and NAD+

  • Measurement of enzyme activity before and after deacetylation

  This approach directly demonstrates the impact of acetylation status on activity without altering expression. Previous studies showed that in vitro deacetylation increased SOD2 specific activity by approximately 35% .

How can researchers investigate the species-specific differences in SOD2 acetylation sites and their functional consequences?

The research literature indicates potential species-specific differences in SOD2 acetylation sites that require specialized approaches to investigate:

• Comparative Mass Spectrometry Analysis:

  • Analysis of SOD2 acetylation sites across human, mouse, and other model organisms

  • Quantitative assessment of acetylation at different lysine residues

  • Correlation with deacetylase activity and oxidative stress conditions

  Previous studies identified different primary acetylation sites: K68 in human cells versus K53/K89 or K122 in mouse cells . This necessitates species-appropriate antibodies and detection methods.

• Cross-Species Mutational Analysis:

  • Creation of site-specific mutations at equivalent positions across species

  • Expression in homologous or heterologous systems

  • Evaluation of functional consequences on enzymatic activity and cellular responses

  Interestingly, while arginine substitution at K53/89 or K122 increased SOD2 activity in mouse models, K68R mutation in human SOD2 did not enhance activity , suggesting that K68 may have additional structural contributions beyond serving as an acetylation site.

• Stress Response Comparison:

  • Examination of how oxidative stress affects SOD2 acetylation patterns across species

  • Evaluation of SIRT3 regulation and activity in different organisms

  • Assessment of downstream consequences on mitochondrial function and cell survival

  Species-specific tools, including the human-specific Acetyl-SOD2 (K68) Recombinant Monoclonal Antibody, are essential for these comparative studies .

What methodological approaches can address the conflicting roles of SOD2 in different disease stages?

The paradoxical roles of SOD2 in disease progression, particularly in cancer (tumor suppressor in early stages versus potential promoter in late stages), require sophisticated experimental designs:

• Stage-Specific Analysis:

  • Temporal analysis of SOD2 expression and acetylation across disease progression

  • Comparison between primary tumors and metastatic lesions from the same patients

  • Correlation with clinical outcomes and treatment responses

  Studies have observed increased SOD2 and HIF2α in metastatic lesions compared to matched primary tumors , suggesting stage-specific changes in SOD2 function.

• Conditional Expression Systems:

  • Inducible expression of SOD2 and acetylation variants at different disease stages

  • Temporal control of SIRT3 activity in disease models

  • Assessment of differential impacts on disease progression and treatment response

  These approaches can help determine whether the timing of SOD2 activity modulation critically affects outcomes.

• Combined Activity Assessment:

  • Simultaneous measurement of both dismutase and peroxidase activities of SOD2

  • Correlation with acetylation status determined by the Acetyl-SOD2 (K68) antibody

  • Evaluation of the balance between these activities in different cellular contexts

  Recent research suggests acetylated SOD2 may gain peroxidase activity while losing dismutase activity , potentially explaining its dual roles in pathophysiology.

The methodological integration of these approaches, enabled by specific tools like the Acetyl-SOD2 (K68) Recombinant Monoclonal Antibody, will help resolve the apparent contradictions in SOD2's role across different disease contexts and stages.