SIRT2 Antibody, Biotin conjugated

What is SIRT2 and why is it an important research target?

SIRT2 (Sirtuin 2) is a NAD-dependent protein deacetylase encoded by the SIRT2 gene in humans. The protein is approximately 43.2 kilodaltons in mass and plays critical roles in multiple biological processes. SIRT2 deacetylates internal lysines on histones, alpha-tubulin, and numerous other proteins including key transcription factors . Its importance as a research target stems from its involvement in cell cycle control, genomic integrity maintenance, microtubule dynamics regulation, cell differentiation, metabolic networks, and autophagy . Recent research has also implicated SIRT2 in cancer progression, particularly in promoting lung cancer metastasis through deacetylation of extracellular proteins such as ITGB3 and collagens . The protein's diverse functions make it a compelling target for researchers studying cellular regulation and disease mechanisms.

What distinguishes biotin-conjugated SIRT2 antibodies from unconjugated variants?

Biotin-conjugated SIRT2 antibodies differ from their unconjugated counterparts primarily in their enhanced detection capabilities. The biotin conjugation provides several methodological advantages: it enables signal amplification through the strong biotin-streptavidin interaction, improves sensitivity in detection systems, and offers versatility in experimental design . Unlike unconjugated antibodies, biotin-conjugated variants can be readily detected using streptavidin coupled to various reporter molecules such as enzymes, fluorophores, or gold particles without requiring species-specific secondary antibodies. This characteristic makes them particularly valuable in multi-labeling experiments, where differentiating between multiple primary antibodies from the same host species would otherwise be challenging . Additionally, the biotin-conjugated format generally exhibits greater stability and provides consistent performance across various detection systems compared to direct enzyme or fluorophore conjugation.

How does SIRT2's subcellular localization influence experimental design when using biotin-conjugated antibodies?

SIRT2's subcellular localization primarily in the cytoplasm significantly impacts experimental design when using biotin-conjugated antibodies . This localization requires careful consideration of cell permeabilization protocols during immunocytochemistry and immunohistochemistry procedures. For effective detection, researchers should implement appropriate fixation methods (such as 4% paraformaldehyde or methanol) followed by detergent-based permeabilization (typically with 0.1-0.5% Triton X-100 or 0.05-0.2% saponin) to ensure antibody access to cytoplasmic compartments without disrupting cellular architecture . When designing co-localization studies, researchers should consider potential steric hindrance from the biotin molecule, which might affect binding to SIRT2 in protein-dense cytoplasmic regions. Additionally, when interpreting results, it's essential to account for SIRT2's reported translocation between cellular compartments under specific conditions, such as during cell cycle progression or cellular stress responses . For optimal visualization, streptavidin conjugates with appropriate spectral properties should be selected to avoid interference from autofluorescence in cytoplasmic regions.

What are the optimal conditions for Western blot applications using biotin-conjugated SIRT2 antibodies?

For optimal Western blot results using biotin-conjugated SIRT2 antibodies, several methodological considerations should be implemented. Based on manufacturer specifications, researchers should use a dilution range of 1:300-1:5000 for the primary antibody incubation . The most effective protocol includes:

• Sample preparation: Use RIPA buffer supplemented with deacetylase inhibitors (such as nicotinamide and trichostatin A) to preserve SIRT2's native acetylation state.

• Gel separation: Employ 10-12% SDS-PAGE gels for optimal resolution of the 43.2 kDa SIRT2 protein.

• Transfer conditions: Use PVDF membranes (rather than nitrocellulose) for better protein retention and signal strength with biotin-conjugated antibodies.

• Blocking: Block with 5% BSA in TBS-T rather than milk-based blockers, as milk contains biotin that may increase background.

• Detection system: Use streptavidin-HRP conjugates at 1:10,000-1:20,000 dilution for detection, followed by enhanced chemiluminescence visualization.

• Controls: Include positive controls from tissues known to express SIRT2 (brain tissue extracts are recommended) and negative controls using blocking peptides to confirm specificity .

This methodology maximizes sensitivity while minimizing background, enabling reliable detection of SIRT2 across human, mouse, and rat samples, with predicted reactivity extending to dog, cow, sheep, pig, horse, and rabbit models .

How should researchers optimize immunohistochemistry protocols for SIRT2 detection using biotin-conjugated antibodies?

For immunohistochemistry applications using biotin-conjugated SIRT2 antibodies, researchers should implement a carefully optimized protocol to ensure specific staining while minimizing background. Based on manufacturer recommendations and research applications, the following methodology is advised:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections of 4-6 μm thickness. For optimal epitope preservation, employ heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes.

• Endogenous biotin blocking: This step is crucial when using biotin-conjugated primary antibodies. Apply an avidin/biotin blocking kit before the primary antibody incubation to neutralize endogenous biotin in tissues, particularly in biotin-rich samples like liver, kidney, and breast tissues.

• Peroxidase quenching: Incubate sections with 0.3% hydrogen peroxide in methanol for 15-30 minutes to block endogenous peroxidase activity.

• Antibody dilution: Use the biotin-conjugated SIRT2 antibody at a dilution of 1:200-1:400 as recommended , and incubate overnight at 4°C in a humidified chamber.

• Detection system: For visualization, utilize streptavidin-HRP conjugates followed by DAB (3,3'-diaminobenzidine) substrate. This approach provides strong signal amplification while maintaining low background.

• Counterstaining: Use hematoxylin for nuclear counterstaining, with careful timing to prevent masking of cytoplasmic SIRT2 signals.

This protocol has been validated for detecting SIRT2 in mouse and rat tissues, with expected reactivity in human samples based on the antibody's immunogen sequence derived from human SIRT2 .

What considerations should be made when designing flow cytometry experiments with biotin-conjugated SIRT2 antibodies?

When designing flow cytometry experiments with biotin-conjugated SIRT2 antibodies, researchers should implement several critical methodological considerations:

• Cell preparation: Since SIRT2 is primarily localized in the cytoplasm , effective permeabilization is essential. Use a gentle permeabilization buffer containing 0.1% saponin or 0.1% Triton X-100 after fixation with 2-4% paraformaldehyde to maintain cellular integrity while allowing antibody access.

• Blocking strategy: Implement a dual blocking approach - first block Fc receptors using 1-2% normal serum from the same species as the secondary reagent, then perform avidin/biotin blocking to minimize background from endogenous biotin.

• Titration optimization: Despite manufacturer recommendations, each experimental system requires antibody titration (typically starting at 1:100 and creating a dilution series) to determine optimal signal-to-noise ratio for the specific cell types under investigation.

• Detection system: Use fluorophore-conjugated streptavidin (such as streptavidin-PE, streptavidin-APC, or streptavidin-Alexa Fluor conjugates) at a concentration of 0.1-0.5 μg/ml. Select fluorophores with emission spectra that avoid overlap with other markers in multi-color panels.

• Compensation controls: Include single-stained controls for each fluorophore to establish proper compensation matrices, particularly important when using bright fluorophores like PE with biotin-streptavidin systems.

• Validation controls: Include appropriate biological controls - SIRT2 knockdown or knockout cells as negative controls, and cells with confirmed SIRT2 overexpression as positive controls .

By implementing these methodological considerations, researchers can achieve reliable quantification of SIRT2 expression in different cell populations while minimizing artifacts and background signal.

How can biotin-conjugated SIRT2 antibodies be utilized to investigate SIRT2's role in cancer metastasis?

Biotin-conjugated SIRT2 antibodies offer sophisticated approaches for investigating SIRT2's emerging role in cancer metastasis, particularly in lung cancer where secreted SIRT2 has been implicated in promoting metastatic progression . A comprehensive methodological approach includes:

• Secretome analysis: Employ biotin-conjugated SIRT2 antibodies in immunoprecipitation followed by mass spectrometry to identify SIRT2-interacting proteins in conditioned media from cancer cells and tumor-associated macrophages. This technique can reveal novel extracellular substrates beyond the already identified ITGB3 and collagens .

• In vitro metastasis models: Utilize the antibodies in transwell migration and invasion assays, applying them to neutralize extracellular SIRT2 activity. Measure changes in cancer cell invasive capacity by comparing results with appropriate controls, including isotype-matched biotin-conjugated antibodies.

• Co-localization studies: Perform dual immunofluorescence using biotin-conjugated SIRT2 antibodies with fluorescently-labeled potential substrates, enabling visualization of SIRT2-substrate interactions in the tumor microenvironment using confocal microscopy.

• Acetylation status assessment: Develop a quantitative immunoassay using biotin-conjugated SIRT2 antibodies alongside acetylation-specific antibodies to measure the acetylation status of ITGB3-K416 and other potential substrates in patient samples, correlating findings with metastatic progression .

• Autophagic secretion pathway: Investigate the role of autophagy in SIRT2 secretion by using the antibodies to track SIRT2 localization in autophagosome structures, combining with markers such as LC3 and ATG7 to validate the secretory mechanism identified in previous research .

This integrated approach enables researchers to comprehensively characterize SIRT2's extracellular functions in the tumor microenvironment and identify potential therapeutic targets for preventing metastasis.

What methodological approaches can resolve contradictory data regarding SIRT2's subcellular distribution and translocation?

To address contradictory findings regarding SIRT2's subcellular distribution and potential translocation between compartments, researchers should implement a multi-faceted methodological approach utilizing biotin-conjugated SIRT2 antibodies:

• Subcellular fractionation validation: Perform rigorous biochemical fractionation followed by Western blotting using biotin-conjugated SIRT2 antibodies to quantitatively assess SIRT2 distribution across cytoplasmic, nuclear, mitochondrial, and membrane fractions. Include well-established markers for each compartment (e.g., GAPDH for cytoplasm, Lamin B1 for nucleus) to confirm fractionation quality .

• Live-cell imaging: Combine biotin-conjugated SIRT2 antibody staining with membrane-permeable streptavidin-fluorophore conjugates in live-cell applications to monitor SIRT2 translocation in response to stimuli such as oxidative stress, nutrient deprivation, or cell cycle progression. This approach minimizes artifacts associated with fixation methods.

• Super-resolution microscopy: Implement techniques such as STORM or PALM using biotin-conjugated SIRT2 antibodies and streptavidin-fluorophore pairs to achieve nanometer-scale resolution of SIRT2 localization, revealing distribution patterns not detectable with conventional microscopy.

• Proximity ligation assays: Combine biotin-conjugated SIRT2 antibodies with antibodies against compartment-specific proteins to detect in situ proximity (typically <40 nm), providing functional evidence of SIRT2's presence in specific subcellular locations.

• Systematic epitope mapping: When contradictory results emerge, assess whether different antibodies recognize distinct SIRT2 epitopes that might be differentially accessible in various cellular compartments or affected by post-translational modifications.

By integrating these complementary approaches, researchers can definitively establish SIRT2's true subcellular distribution patterns, reconcile seemingly contradictory data in the literature, and potentially identify previously unrecognized regulatory mechanisms controlling SIRT2 localization and function .

How can researchers investigate the role of SIRT2 in the autophagy-dependent secretion pathway using biotin-conjugated antibodies?

To investigate SIRT2's role in the autophagy-dependent secretion pathway, researchers can implement a comprehensive methodological approach using biotin-conjugated SIRT2 antibodies:

• Autophagosome co-localization analysis: Perform dual immunofluorescence staining using biotin-conjugated SIRT2 antibodies with streptavidin-fluorophore detection systems, alongside antibodies against autophagy markers such as LC3, ATG7, and p62. Analyze co-localization using confocal microscopy with quantitative colocalization coefficients (Pearson's or Mander's) to measure the degree of SIRT2 association with autophagic structures under basal conditions and following TLR2/TLR4 stimulation .

• Proximity-based protein interaction studies: Implement proximity ligation assays (PLA) using biotin-conjugated SIRT2 antibodies together with antibodies against autophagy-related proteins to visualize and quantify molecular interactions within intact cells, providing spatial resolution of <40 nm to confirm direct associations.

• Secretion pathway tracking: Employ the bead halo assay, previously validated for studying protein secretion, using biotin-conjugated SIRT2 antibodies to detect extracellular SIRT2. This method can be modified by introducing inhibitors of conventional and unconventional secretion pathways to distinguish the specific route of SIRT2 export .

• Genetic manipulation validation: Combine biotin-conjugated SIRT2 antibody detection with genetic approaches, such as CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of key autophagy genes (ATG5, ATG7, BECN1) to confirm the dependency of SIRT2 secretion on the autophagy machinery. Quantify changes in extracellular SIRT2 levels using ELISA or Western blot of conditioned media .

• TLR signaling pathway dissection: As TLR4/TLR2 stimulation has been shown to trigger SIRT2 secretion, use biotin-conjugated SIRT2 antibodies to monitor SIRT2 trafficking following stimulation with specific ligands, incorporating inhibitors of TRAF6's E3 ligase activity to validate its role as a checkpoint in SIRT2 secretion .

This integrated approach provides a robust methodological framework for elucidating the molecular mechanisms governing SIRT2's unconventional secretion via the autophagy pathway, advancing our understanding of how this primarily intracellular deacetylase functions in the extracellular environment.

What quality control measures should be implemented to validate biotin-conjugated SIRT2 antibody specificity?

To ensure experimental rigor when using biotin-conjugated SIRT2 antibodies, researchers should implement a comprehensive quality control validation framework:

• Genetic validation controls: Test antibody specificity using SIRT2 knockout or knockdown models (CRISPR/Cas9-edited cell lines or siRNA-treated cells) compared to wild-type controls. Complete absence or significant reduction of signal in these models provides strong evidence of specificity .

• Epitope blocking experiments: Pre-incubate the biotin-conjugated SIRT2 antibody with excess immunizing peptide (the synthetic peptide derived from human SIRT2 residues 261-360/389 used as the immunogen) prior to application in experimental procedures. Signal abolishment confirms epitope-specific binding.

• Cross-reactivity assessment: Test the antibody against recombinant proteins of other sirtuin family members (SIRT1, SIRT3-7) to confirm absence of cross-reactivity with structurally similar proteins, particularly important given the conserved catalytic domain among sirtuins.

• Orthogonal method comparison: Validate findings by comparing results obtained with the biotin-conjugated SIRT2 antibody against those generated using alternative SIRT2 antibodies recognizing different epitopes, or with orthogonal techniques such as mass spectrometry.

• Species reactivity verification: Systematically test antibody performance across predicted reactive species (human, mouse, rat, dog, cow, sheep, pig, horse, rabbit) using appropriate positive control tissues/cells from each species. Western blot analysis should confirm specific detection at the expected molecular weight (43.2 kDa).

• Lot-to-lot consistency testing: When receiving new antibody lots, perform side-by-side comparison with previous lots using standardized positive control samples to ensure consistent performance across manufacturing batches.

Implementing these rigorous validation measures ensures experimental reliability and facilitates meaningful interpretation of results obtained with biotin-conjugated SIRT2 antibodies across diverse experimental systems.

How can researchers troubleshoot high background issues when using biotin-conjugated SIRT2 antibodies in immunohistochemistry?

When encountering high background issues with biotin-conjugated SIRT2 antibodies in immunohistochemistry, researchers should implement a systematic troubleshooting approach:

• Endogenous biotin blocking optimization: Endogenous biotin is the primary source of background with biotin-conjugated antibodies. Implement a sequential avidin-biotin blocking step using commercial kits, with extended incubation times (30-45 minutes for each component) for biotin-rich tissues such as liver, kidney, and brain. For particularly problematic samples, consider adding free streptavidin (10-20 μg/ml) in the blocking solution to saturate endogenous biotin sites .

• Enhanced tissue preparation: Implement dual fixation protocols combining brief (15-20 minute) paraformaldehyde fixation followed by acetone post-fixation to preserve antigen accessibility while maintaining tissue architecture. Optimize antigen retrieval conditions by testing multiple buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0) and heating methods (microwave, pressure cooker, water bath) to determine optimal conditions for SIRT2 epitope exposure.

• Blocking buffer optimization: Prepare a multi-component blocking solution containing:

  • 5-10% serum from the species of the streptavidin conjugate

  • 1% BSA (IgG-free, protease-free)

  • 0.1-0.3% Triton X-100 or 0.05% saponin for improved penetration

  • 0.1% cold water fish skin gelatin to reduce non-specific binding
    Extend blocking time to 2 hours at room temperature or overnight at 4°C.

• Antibody dilution matrix: Create a dilution matrix testing the biotin-conjugated SIRT2 antibody at multiple concentrations (1:100, 1:200, 1:400, 1:800) against varying concentrations of streptavidin-HRP (1:100, 1:500, 1:1000, 1:2000) to identify the optimal signal-to-noise ratio combination .

• Absorption controls: Pre-absorb the biotin-conjugated antibody with tissue homogenates from species known to lack or express minimal SIRT2 to remove potentially cross-reactive antibodies from the preparation.

By systematically implementing these troubleshooting strategies, researchers can achieve clean, specific staining patterns with biotin-conjugated SIRT2 antibodies, enabling accurate localization and quantification of SIRT2 expression in tissue sections.

What methodological considerations address potential artifacts when studying SIRT2-mediated deacetylation of extracellular proteins?

Investigating SIRT2-mediated deacetylation of extracellular proteins presents unique methodological challenges that require specialized approaches to prevent artifacts and ensure data validity:

• Acetylation preservation strategy: Implement a comprehensive acetylation preservation protocol during sample collection and processing by including a cocktail of deacetylase inhibitors (1-5 mM nicotinamide, 1-5 μM trichostatin A, and 10 mM sodium butyrate) in all buffers. Additionally, maintain samples at 4°C throughout processing to minimize enzymatic activity that could alter acetylation status prior to analysis .

• Extracellular versus intracellular SIRT2 discrimination: Develop a sequential extraction protocol that separately isolates extracellular matrix/secreted proteins from cellular components before immunoprecipitation with biotin-conjugated SIRT2 antibodies. This approach prevents contamination with intracellular SIRT2 that could lead to false positive interactions or deacetylation events.

• In vitro validation system: Establish a reconstituted in vitro system using purified components:

  • Recombinant human SIRT2 protein

  • Synthetically acetylated substrate peptides from ITGB3-K416 and collagen

  • NAD+ cofactor at physiologically relevant concentrations
    This controlled system allows confirmation of direct deacetylation without cellular confounding factors .

• Mass spectrometry validation: Implement quantitative MS approaches (SILAC or TMT labeling) to measure changes in acetylation stoichiometry at specific lysine residues of candidate substrates, providing direct biochemical evidence of SIRT2-mediated deacetylation that complements antibody-based detection methods.

• Proximity-based interaction mapping: Utilize techniques such as BioID or APEX2 proximity labeling to identify proteins in close physical proximity to extracellular SIRT2, confirming candidate substrates identified through other means and potentially discovering novel interaction partners.

• Microenvironment reconstruction: Develop 3D culture systems incorporating extracellular matrix components to recapitulate the physiological environment where SIRT2-substrate interactions occur, addressing the potential artifact of non-physiological interactions observed in simplified 2D cultures .

By implementing these methodological considerations, researchers can confidently investigate SIRT2's extracellular deacetylation functions while minimizing artifacts that might otherwise confound interpretation of experimental results.

How might biotin-conjugated SIRT2 antibodies facilitate investigation of therapeutic targeting of the SIRT2 secretion pathway?

Biotin-conjugated SIRT2 antibodies offer sophisticated methodological approaches for investigating therapeutic interventions targeting the SIRT2 secretion pathway, particularly in cancer contexts where extracellular SIRT2 promotes metastasis . A comprehensive research strategy includes:

• High-throughput screening platform: Develop a cell-based screening system using macrophages or cancer cells where biotin-conjugated SIRT2 antibodies combined with fluorescent streptavidin detect secreted SIRT2 in culture supernatants. This platform enables screening of compound libraries to identify inhibitors of SIRT2 secretion through the autophagy-dependent pathway, with quantitative readouts via fluorescence intensity measurements.

• TRAF6-focused intervention assessment: Given TRAF6's identified role as a checkpoint for SIRT2 secretion , establish an assay combining biotin-conjugated SIRT2 antibodies with proximity ligation technology to monitor TRAF6-SIRT2 interactions in situ. This methodology allows evaluation of compounds designed to disrupt this specific interaction without affecting other TRAF6 functions.

• Therapeutic antibody development pipeline: Adapt the biotin-conjugated SIRT2 antibody framework to develop therapeutic antibodies that neutralize extracellular SIRT2 activity. Implement a systematic validation approach:

  • In vitro deacetylation assays using acetylated ITGB3 substrates

  • Cell-based migration/invasion assays

  • Ex vivo tissue explant systems

  • In vivo metastasis models with antibody treatment

• Extracellular vesicle (EV) characterization: Apply biotin-conjugated SIRT2 antibodies in immunogold electron microscopy to determine if SIRT2 is packaged within EVs during secretion. This approach enables assessment of therapeutic strategies targeting EV biogenesis as an indirect means of preventing SIRT2 secretion.

• Biomarker development: Develop sensitive immunoassays using biotin-conjugated SIRT2 antibodies to quantify circulating SIRT2 levels in patient serum, correlating with disease progression and response to therapy. This approach supports patient stratification for targeted anti-SIRT2 interventions and provides a monitoring tool for treatment efficacy .

This integrated approach establishes a robust methodological framework for therapeutic development targeting the SIRT2 secretion pathway, potentially leading to novel interventions for metastasis prevention in cancer patients.

What methodological approaches can elucidate SIRT2's role in the cell cycle and genomic stability?

To investigate SIRT2's complex roles in cell cycle regulation and genomic stability, researchers should implement a comprehensive methodological framework utilizing biotin-conjugated SIRT2 antibodies:

• Cell cycle-specific SIRT2 localization profiling: Synchronize cells at different cell cycle phases (G1, S, G2, M) using established methods (double thymidine block, nocodazole treatment, etc.), then perform immunofluorescence microscopy and subcellular fractionation with biotin-conjugated SIRT2 antibodies to track SIRT2 localization changes throughout the cell cycle. Combine with co-localization studies using markers for specific subcellular structures associated with mitotic progression .

• Chromatin immunoprecipitation sequencing (ChIP-seq): Adapt standard ChIP protocols for use with biotin-conjugated SIRT2 antibodies to map SIRT2 binding across the genome at different cell cycle stages. Streptavidin-conjugated magnetic beads provide efficient capture of biotin-antibody-SIRT2-DNA complexes, enabling identification of cell cycle-regulated genomic binding sites at transcriptional start sites and enhancers of active genes .

• Deacetylase activity time-course analysis: Develop a quantitative assay measuring SIRT2 enzymatic activity toward key substrates throughout the cell cycle, combining immunoprecipitation using biotin-conjugated SIRT2 antibodies with activity assays utilizing fluorescent or bioluminescent deacetylase substrates. This approach reveals temporal regulation of SIRT2 enzymatic function.

• APC/C activity correlation studies: Implement methodologies to assess how SIRT2-mediated deacetylation of CDC20 and FZR1 affects APC/C ubiquitin ligase activity during mitotic progression. Use biotin-conjugated SIRT2 antibodies to immunoprecipitate complexes, followed by in vitro ubiquitination assays to measure functional consequences of SIRT2 activity .

• Microtubule stress response analysis: Establish live-cell imaging protocols using biotin-conjugated SIRT2 antibodies with cell-permeable fluorescent streptavidin to track SIRT2 dynamics during antephase checkpoint activation in response to microtubule stress agents. Correlate SIRT2 localization and activity with chromosome segregation outcomes to elucidate its role in preventing precocious mitotic entry .

This integrated approach enables comprehensive characterization of SIRT2's multifaceted functions in maintaining genomic integrity throughout the cell cycle, potentially revealing novel therapeutic targets for conditions characterized by genomic instability.

How can biotin-conjugated SIRT2 antibodies be utilized in multiplexed imaging approaches to study SIRT2 in complex tissue environments?

Biotin-conjugated SIRT2 antibodies offer significant advantages for multiplexed imaging applications in complex tissue environments due to their compatibility with diverse detection systems. A comprehensive methodological framework includes:

• Cyclic immunofluorescence (CycIF) protocol development: Establish a CycIF protocol using biotin-conjugated SIRT2 antibodies with different streptavidin-fluorophore conjugates in sequential staining rounds. This approach allows co-visualization of SIRT2 with up to 30-40 additional proteins in the same tissue section through iterative staining, imaging, and signal removal cycles. Specific methodology includes:

  • Initial application of biotin-conjugated SIRT2 antibody (1:200-1:400 dilution)

  • Detection with spectrally distinct streptavidin conjugates

  • Image acquisition

  • Chemical inactivation of fluorophores

  • Repetition with additional markers

• Mass cytometry adaptation: Modify standard immunohistochemistry protocols for use with biotin-conjugated SIRT2 antibodies in mass cytometry (CyTOF) applications by using streptavidin conjugated to rare earth metals. This enables simultaneous detection of SIRT2 alongside 40+ additional markers in tissue sections or dissociated cells, providing unprecedented phenotypic detail of SIRT2-expressing cells.

• Spatial transcriptomics integration: Combine biotin-conjugated SIRT2 antibody staining with spatial transcriptomics techniques using sequential immunofluorescence and in situ hybridization. This approach correlates SIRT2 protein expression with transcriptional profiles in specific tissue regions, revealing potential regulatory relationships in their native context.

• Digital spatial profiling optimization: Develop a protocol using biotin-conjugated SIRT2 antibodies with oligonucleotide-tagged streptavidin for digital spatial profiling. This method enables precise quantification of SIRT2 protein levels in user-defined regions of interest within heterogeneous tissues, with data output as spatially resolved protein expression maps.

• 3D tissue clearing compatibility testing: Validate compatibility of biotin-conjugated SIRT2 antibodies with various tissue clearing techniques (CLARITY, iDISCO, CUBIC) to enable whole-organ imaging with single-cell resolution. Optimize penetration by testing different antibody concentrations (1:100-1:500) and extended incubation times (3-7 days) for thick tissue sections.

This integrated approach establishes a methodological framework for comprehensively mapping SIRT2 expression and function in complex tissues, enabling new insights into its context-specific roles across different physiological and pathological states.