sirt5 Antibody

What is SIRT5 and what are its primary functions in cellular metabolism?

SIRT5 is a member of the sirtuin family of NAD+-dependent protein deacylases that primarily functions as a desuccinylase, demalonylase, and deglutarylase. It plays a critical role in regulating both mitochondrial and cytosolic protein post-translational modifications. SIRT5 has been identified as a global regulator of lysine malonylation, providing a mechanism for regulation of energetic flux through glycolysis . In tissue-specific analyses, SIRT5 expression is particularly prominent in metabolically active organs, with highest protein malonylation detected in kidney and liver tissues, while protein succinylation appears most abundant in heart tissue .

To effectively study SIRT5 function, researchers should consider using antibodies that detect both mitochondrial and cytosolic forms, as SIRT5 has been confirmed to localize to both cellular compartments . The protein exhibits a molecular weight of approximately 33-35 kDa when detected by western blot analysis .

How do I select the appropriate SIRT5 antibody for my experimental applications?

Selection of a SIRT5 antibody should be guided by your specific experimental application and target species. Based on validated research applications, consider the following methodological approach:

• For Western Blot analysis: Select antibodies specifically validated for this application, such as those demonstrating specific detection at approximately 33-35 kDa in human liver tissue lysates . Recommended dilutions typically range from 1:2000 to 1:10000, but optimization for your specific sample type is advised .

• For Immunohistochemistry: Choose antibodies validated in fixed paraffin-embedded tissue sections. For example, anti-SIRT5 antibodies have been successfully used at concentrations of 5-15 μg/mL for human liver sections with specific staining localized to hepatocyte cytoplasm . For IHC applications, a dilution range of 1:250 to 1:1000 is typically recommended .

• For cross-reactivity considerations: Verify specificity by checking cross-reactivity with other sirtuin family members. High-quality antibodies show minimal cross-reactivity (<1%) with related proteins such as SIRT1, SIRT2, and SIRT6 .

What are the recommended protocols for detecting SIRT5 in tissue samples?

For optimal detection of SIRT5 in tissue samples, follow these methodological guidelines:

For immunohistochemistry in paraffin-embedded sections:

• Perform antigen retrieval using TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0 may be used)

• Incubate with primary SIRT5 antibody at 10 μg/mL overnight at 4°C

• Apply appropriate secondary antibody system (e.g., Anti-Sheep HRP-DAB)

• Counterstain with hematoxylin to visualize cellular structures

For western blot detection:

• Prepare protein lysates from tissue samples under reducing conditions

• Separate proteins using SDS-PAGE and transfer to PVDF membrane

• Block membrane appropriately (follow specific buffer recommendations, such as Immunoblot Buffer Group 8)

• Probe with SIRT5 antibody at 1 μg/mL

• Apply HRP-conjugated secondary antibody appropriate to the host species of primary antibody

• Develop using chemiluminescence detection system

Positive controls should include human liver tissue, where SIRT5 is abundantly expressed and detected at approximately 33-35 kDa .

How can I assess SIRT5 enzymatic activity in cellular models?

Assessing SIRT5 enzymatic activity requires methodologies that detect specific post-translational modifications regulated by this enzyme. Based on research findings, the following approaches are recommended:

Global protein acylation analysis:

• Prepare cellular lysates from experimental and control samples

• Perform western blot analysis using antibodies specific for succinyllysine, malonyllysine, or glutaryllysine modifications

• Compare modification levels between wild-type and SIRT5-deficient or variant samples

• For enhanced detection of succinylation, cells can be treated with dimethyl succinate-ester (a cell-permeable succinate analogue) to increase substrate availability

Subcellular localization of acylation:

• Perform immunofluorescence staining using anti-succinyllysine antibodies

• Co-stain with mitochondrial markers (e.g., Mitotracker)

• Analyze co-localization to determine mitochondrial versus cytosolic distribution of modified proteins

Research has demonstrated that SIRT5 deficiency results in significantly increased global succinyllysine levels, with modifications detected in both mitochondrial and cytosolic compartments . This approach allows for functional assessment of SIRT5 activity without requiring purified enzyme.

What are the key considerations when studying SIRT5 variants associated with disease?

When investigating SIRT5 variants associated with disease states, several methodological considerations should be addressed:

Protein stability assessment:

• Analyze protein levels of SIRT5 variants compared to wild-type using validated antibodies

• Use multiple antibodies generated against distinct antigens to rule out artifactual reductions due to altered antibody affinity

• Include controls for total mitochondrial content (e.g., Complex V/ATP5A subunit) to normalize mitochondrial protein levels

Enzymatic activity analysis:

• For cellular models, assess global protein acylation status using antibodies against succinyllysine, malonyllysine, and glutaryllysine

• For biochemical assays, measure NAD+-dependent desuccinylase activity using purified recombinant enzymes

• Test enzymatic function under varying substrate and NAD+ concentrations to identify conditions where variant enzymes show deficits

Research on SIRT5 variants P114T and L128V from mitochondrial disease patients revealed that these variants exhibited:

• Reduced protein stability

• Decreased desuccinylase activity (30% reduction with standard NAD+ levels, 50% reduction under limiting NAD+ conditions)

• Increased global protein succinylation in patient fibroblasts

These methodologies allow for comprehensive functional characterization of SIRT5 variants with potential pathological significance.

How can I differentiate between mitochondrial and cytosolic SIRT5 functions in cellular models?

Differentiating between mitochondrial and cytosolic SIRT5 functions requires specific subcellular fractionation and localization techniques:

Subcellular fractionation approach:

• Isolate mitochondrial and cytosolic fractions using established differential centrifugation protocols

• Analyze protein malonylation and succinylation levels in each fraction separately

• Compare patterns between wild-type and SIRT5-deficient samples

Immunofluorescence co-localization:

• Perform double immunofluorescence using SIRT5 antibodies and mitochondrial markers

• Analyze the degree of co-localization to determine mitochondrial versus cytosolic distribution

• For functional analysis, use antibodies against acylated proteins to visualize the subcellular distribution of SIRT5 substrates

Research has demonstrated that SIRT5 regulates both mitochondrial and cytosolic protein malonylation and succinylation, with tissue-specific patterns of modification . In liver and kidney, where SIRT5 expression is highest, protein malonylation shows significant increases in SIRT5-deficient models, while succinylation is most prominent in heart tissue .

What controls should be included when validating SIRT5 antibody specificity?

Rigorous validation of SIRT5 antibody specificity requires several methodological controls:

Positive controls:

• Human liver tissue (high endogenous SIRT5 expression)

• Cell lines with confirmed SIRT5 expression (HeLa, HEK-293, L02, LNCaP, K-562)

Negative controls:

• SIRT5 knockout or knockdown samples (if available)

• Pre-absorption with immunizing peptide

• Isotype control antibody matching the SIRT5 antibody host species

Cross-reactivity assessment:

• Test reactivity with recombinant SIRT family members (SIRT1, SIRT2, SIRT6)

• Verify minimal cross-reactivity (<1%) in direct ELISA assays

Multiple antibody validation:

• When studying SIRT5 variants, use at least two antibodies generated against distinct antigenic regions to confirm specificity

• Compare antibody detection patterns in western blot, IHC, and immunofluorescence applications

Following these validation steps ensures that observed signals are specific to SIRT5 and not due to cross-reactivity with related proteins or non-specific binding.

How do I optimize detection of SIRT5-regulated protein modifications?

Optimizing detection of SIRT5-regulated protein modifications requires careful consideration of experimental conditions:

For enhanced detection of protein succinylation:

• Treat cells with dimethyl succinate-ester to increase intracellular succinyl-CoA levels and enhance protein succinylation

• Use anti-succinyllysine antibodies with demonstrated specificity

• For western blot, optimize primary antibody concentration and incubation conditions

• For immunofluorescence, co-stain with mitochondrial markers to assess subcellular localization

For analysis of multiple acylation types:

• Perform parallel immunoblots using antibodies against succinyllysine, malonyllysine, and glutaryllysine

• Include both SIRT5-deficient and wild-type samples for comparison

• Normalize acylation signals to total protein loading

Research has shown that SIRT5 deficiency results in most prominent increases in protein succinylation, while effects on malonylation and glutarylation may be more subtle or tissue-specific . The selection of appropriate acylation-specific antibodies is critical for accurate assessment of SIRT5 activity.

What are the potential pitfalls when interpreting SIRT5 antibody results across different tissue types?

When working with SIRT5 antibodies across different tissue types, researchers should be aware of several potential interpretational pitfalls:

Tissue-specific expression levels:

• SIRT5 expression varies significantly across tissues, with highest levels in liver and kidney

• Low-expressing tissues may require more sensitive detection methods or increased antibody concentrations

• Always include positive control tissues (e.g., liver) alongside experimental tissues

Differential acylation patterns:

• Protein succinylation is most abundant in heart tissue, while malonylation is highest in liver and kidney

• The primary SIRT5-regulated modification may differ between tissues

• Test multiple acylation-specific antibodies to comprehensively assess SIRT5 function

Subcellular distribution variations:

• The ratio of mitochondrial to cytosolic SIRT5 may vary between tissue types

• Perform subcellular fractionation or co-localization studies to determine tissue-specific distribution patterns

• Consider that observed phenotypes may reflect compartment-specific SIRT5 functions

Background and non-specific binding:

• Some tissues may exhibit higher background with certain antibody preparations

• Optimize blocking conditions for each tissue type

• Include isotype control antibodies to assess non-specific binding

How can SIRT5 antibodies be used to investigate metabolic pathway regulation?

SIRT5 antibodies can be powerful tools for investigating metabolic pathway regulation through the following methodological approaches:

Identification of SIRT5 substrates in metabolic pathways:

• Perform immunoprecipitation using anti-SIRT5 antibodies to pull down SIRT5-interacting proteins

• Couple with mass spectrometry to identify novel interacting partners

• Validate interactions through reciprocal co-immunoprecipitation experiments

Analysis of enzyme modification status:

• Use antibodies against specific acylations (succinyl, malonyl, glutaryl) to detect modifications on metabolic enzymes

• Compare modification status between wild-type and SIRT5-deficient samples

• Correlate modification status with enzymatic activity measurements

Research has shown that SIRT5 regulates energetic flux through glycolysis by controlling protein malonylation . Additionally, SIRT5 has been demonstrated to activate CPS1 (carbamoyl phosphate synthetase 1) during prolonged fasting , suggesting a role in regulating the urea cycle.

What methodological approaches can resolve contradictions in SIRT5 research findings?

When faced with contradictory findings in SIRT5 research, consider the following methodological approaches:

Antibody validation and standardization:

• Use multiple antibodies generated against different epitopes

• Include appropriate positive and negative controls

• Standardize detection methods across experiments

Conditional experimental models:

• Examine SIRT5 function under different metabolic conditions (fed vs. fasted, glycolytic vs. oxidative)

• Consider that NAD+ levels can fluctuate dramatically in response to altered metabolic conditions, affecting SIRT5 activity

• Test SIRT5 function under varying substrate and NAD+ concentrations to identify condition-dependent effects

Tissue and cell-type specificity:

• Analyze SIRT5 function across multiple tissues and cell types

• Consider that SIRT5 may have tissue-specific roles based on metabolic demands

• Use tissue-specific knockout models when available

Research has demonstrated that SIRT5 variants may show reduced activity (30-50% decrease) under limiting NAD+ conditions, but normal activity under standard conditions . This highlights the importance of considering metabolic context when interpreting SIRT5 function.

How should researchers integrate SIRT5 antibody data with other omics approaches?

For comprehensive understanding of SIRT5 biology, integration of antibody-based data with other omics approaches is essential:

Integration with proteomics:

• Combine SIRT5 immunoprecipitation with mass spectrometry to identify interacting partners

• Use acylation-specific antibodies for enrichment of modified proteins followed by mass spectrometry

• Correlate changes in protein expression with changes in acylation status

Integration with metabolomics:

• Compare metabolite profiles between wild-type and SIRT5-deficient samples

• Correlate changes in protein acylation with alterations in metabolic pathways

• Use stable isotope labeling to track metabolic flux in the presence or absence of SIRT5 activity

Integration with transcriptomics:

• Analyze gene expression changes in response to SIRT5 manipulation

• Identify potential transcriptional effects downstream of SIRT5-mediated metabolic regulation

• Compare transcriptional profiles across different tissues to identify tissue-specific responses

This integrative approach provides a systems-level understanding of SIRT5 function beyond what can be achieved with antibody-based techniques alone. For example, while SIRT5 variants P114T and L128V show reduced protein stability and activity, mouse models with the P114T mutation did not display obvious metabolic abnormalities or neuropathology , suggesting compensatory mechanisms that might be identified through integrated omics approaches.