SIRT1 Antibody, Biotin conjugated

What is SIRT1 and what cellular functions does it regulate?

SIRT1 (silent mating type information regulation 2 homolog 1) is a NAD-dependent protein deacetylase belonging to the sirtuin family of proteins, which are homologs to the yeast Sir2 protein. SIRT1 plays critical roles in numerous cellular processes including DNA damage response (DDR), DNA repair, epigenetic gene silencing, and metabolic regulation. It exerts its functions primarily through deacetylating various target proteins, including histones and transcription factors. SIRT1 has a subcellular localization in both the cytoplasm and nucleus, enabling it to regulate diverse cellular processes in multiple compartments .

SIRT1 has been particularly noted for its role in the DNA damage response pathway, where it rapidly mobilizes to DNA double-strand breaks (DSBs) and works synergistically with other proteins such as SIRT6 to facilitate repair processes. For instance, SIRT1 deacetylates SIRT6 at K33, promoting SIRT6 polymerization and recognition of DSBs, which subsequently enhances chromatin remodeling through deacetylation of H3K9ac and H3K56ac .

What are the technical specifications of SIRT1 Antibody, Biotin Conjugated?

The SIRT1 Antibody, Biotin Conjugated (catalog #bs-2257R-Biotin) is a polyclonal antibody derived from rabbit immunized with KLH-conjugated synthetic peptide from human SIRT1. The immunogen range spans amino acids 551-650/747 of the SIRT1 protein. This antibody has a concentration of 1μg/μl and is purified using Protein A. The storage buffer consists of an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol. For optimal preservation, it should be stored at -20°C, where it remains stable for 12 months .

The antibody demonstrates confirmed reactivity with human, mouse, and dog samples, with predicted reactivity in pig, horse, and rabbit systems. Its biotin conjugation makes it particularly versatile for detection methods utilizing avidin-biotin systems .

What applications is the SIRT1 Antibody, Biotin Conjugated suitable for?

The SIRT1 Antibody, Biotin Conjugated has been validated for multiple applications in molecular and cellular biology research:

The antibody has demonstrated positive Western blot detection in multiple cell lines including HEK-293, HeLa, and MDA-MB-231 cells. For IHC applications, it has successfully detected SIRT1 in human colon cancer tissue and lung cancer tissue .

It is important to note that optimal dilutions may vary depending on the specific experimental conditions and sample types, so preliminary titration experiments are recommended to determine the most suitable working concentration for your particular research application.

How should I optimize Western blot protocols when using SIRT1 Antibody, Biotin Conjugated?

When optimizing Western blot protocols for SIRT1 Antibody, Biotin Conjugated, consider the following methodological approach:

Sample Preparation:

• Extract proteins using a lysis buffer containing protease inhibitors to prevent degradation of SIRT1 (MW: 82 kDa, observed at 110-130 kDa or 80-85 kDa depending on post-translational modifications).

• Include phosphatase inhibitors if phosphorylated forms of SIRT1 are of interest.

• Load 20-50 μg of total protein per lane for optimal detection.

Gel Electrophoresis and Transfer:

• Use 8-10% SDS-PAGE gels for optimal resolution of SIRT1.

• Transfer to PVDF membranes (preferred over nitrocellulose for this antibody).

• Transfer at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol.

Blocking and Antibody Incubation:

• Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature.

• Dilute primary antibody (SIRT1 Antibody, Biotin Conjugated) between 1:1000-1:6000 in blocking buffer.

• Incubate overnight at 4°C with gentle rocking.

• Wash 3 times with TBST, 5 minutes each.

• Incubate with streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature.

• Wash 3 times with TBST, 5 minutes each.

Detection:

• Use enhanced chemiluminescence (ECL) reagents for detection.

• Expected molecular weight: 110-130 kDa or 80-85 kDa bands representing SIRT1.

For troubleshooting, if background is high, increase washing steps or further dilute the antibody. If signal is weak, extend exposure time or use a more sensitive detection system such as ECL Plus or Super Signal West Femto. Always include appropriate positive controls such as HEK-293 or HeLa cell lysates .

What are the recommended protocols for immunohistochemistry using SIRT1 Antibody, Biotin Conjugated?

For optimal immunohistochemistry results with SIRT1 Antibody, Biotin Conjugated, follow this methodological approach:

Tissue Preparation:

• For paraffin-embedded tissues (IHC-P): Cut sections at 4-6 μm thickness.

• For frozen tissues (IHC-F): Cut sections at 5-8 μm thickness and fix in cold acetone.

Antigen Retrieval:

• For paraffin sections: Perform heat-induced epitope retrieval using TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative).

• Heat in a pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 10-20 minutes.

• Allow slides to cool in the retrieval solution for 20 minutes at room temperature.

Staining Protocol:

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes.

• Wash with PBS, 3 times, 5 minutes each.

• Block non-specific binding with 10% normal serum for 30 minutes.

• Apply SIRT1 Antibody, Biotin Conjugated at 1:500-1:2000 dilution in blocking buffer.

• Incubate overnight at 4°C in a humidified chamber.

• Wash with PBS, 3 times, 5 minutes each.

• Apply streptavidin-HRP for 30 minutes at room temperature.

• Wash with PBS, 3 times, 5 minutes each.

• Develop with DAB substrate until desired color intensity is achieved (typically 2-10 minutes).

• Counterstain with hematoxylin, dehydrate, clear, and mount.

Controls and Validation:

• Include positive control tissues (human colon cancer tissue or lung cancer tissue).

• Include a negative control by omitting primary antibody.

• Evaluate SIRT1 staining in both nuclear and cytoplasmic compartments, as SIRT1 can localize to both regions .

For troubleshooting weak staining, consider extending antibody incubation time, optimizing antigen retrieval conditions, or using a more sensitive detection system such as a biotin-streptavidin amplification system.

How can SIRT1 Antibody, Biotin Conjugated be utilized to investigate SIRT1-SIRT6 interactions in DNA damage response?

To investigate the synergistic interaction between SIRT1 and SIRT6 in DNA damage response, SIRT1 Antibody, Biotin Conjugated can be employed in several advanced experimental approaches:

Co-Immunoprecipitation (Co-IP) Studies:

• Induce DNA damage in cells using agents such as etoposide (10 μM, 1 hour) or gamma-irradiation (10 Gy).

• Harvest cells at different time points post-damage (0, 5, 15, 30, 60 minutes).

• Perform immunoprecipitation using SIRT1 Antibody, Biotin Conjugated coupled to streptavidin beads.

• Analyze precipitates for SIRT6 presence by Western blot.

• Compare the interaction kinetics before and after DNA damage induction.

This approach has revealed that SIRT1 directly interacts with SIRT6, as demonstrated by co-immunoprecipitation studies in HEK293 cells where FLAG-SIRT6 interacted with endogenous SIRT1 and vice versa .

Chromatin Immunoprecipitation (ChIP) Assays:

• Induce DSBs at specific genomic loci (e.g., using CRISPR-Cas9 system).

• Perform ChIP using SIRT1 Antibody, Biotin Conjugated to assess SIRT1 recruitment to DSB sites.

• Conduct sequential ChIP (re-ChIP) to determine co-occupancy of SIRT1 and SIRT6 at DSB sites.

• Analyze by qPCR using primers flanking the DSB sites.

Research has shown that upon DNA damage, both SIRT1 and SIRT6 are rapidly mobilized to DSBs, with SIRT1 redistributing on chromatin to deacetylate various repair factors including XPA, NBS1, and Ku70 .

Deacetylation Assays:

• Immunoprecipitate SIRT6 from cells treated with SIRT1 inhibitors (nicotinamide or Ex527).

• Assess SIRT6 acetylation status using pan-acetyl lysine antibodies.

• Focus on K33 acetylation, as research indicates this is a key site deacetylated by SIRT1.

Studies have demonstrated that SIRT6 acetylation levels increase in the presence of SIRT1 inhibitors, and that SIRT1 specifically targets K33ac on SIRT6 .

What methodological approaches can be used to study post-translational modifications of SIRT1 using the biotin-conjugated antibody?

Studying post-translational modifications (PTMs) of SIRT1 requires sophisticated methodological approaches, for which the SIRT1 Antibody, Biotin Conjugated can be adapted:

Tandem Affinity Purification:

• Use SIRT1 Antibody, Biotin Conjugated to capture SIRT1 from cellular lysates.

• Elute purified SIRT1 complexes.

• Perform mass spectrometry analysis to identify PTMs (phosphorylation, acetylation, sumoylation, etc.).

• Compare PTM profiles under different cellular conditions (e.g., normal vs. stressed cells).

Sequential Immunoprecipitation:

• First immunoprecipitation: Use SIRT1 Antibody, Biotin Conjugated to isolate total SIRT1.

• Second immunoprecipitation: Use antibodies specific for PTMs (e.g., anti-phospho, anti-acetyl).

• Analyze the doubly-immunoprecipitated material to quantify specific modified forms of SIRT1.

Two-dimensional Gel Electrophoresis:

• Immunoprecipitate SIRT1 using the biotin-conjugated antibody.

• Separate the precipitated proteins by 2D gel electrophoresis.

• Identify SIRT1 spots using Western blotting.

• Analyze shifts in isoelectric point and molecular weight to detect PTMs.

• Extract spots for mass spectrometry analysis to identify specific modifications.

Research has shown that SIRT1's activity and localization are modulated by various PTMs. For example, SIRT1 deacetylates SIRT6 at K33, but SIRT6 does not appear to affect SIRT1 acetylation levels . Understanding these modification patterns is crucial for elucidating SIRT1's role in diverse cellular processes.

How can I analyze the spatiotemporal dynamics of SIRT1 recruitment to DNA damage sites?

To analyze the spatiotemporal dynamics of SIRT1 recruitment to DNA damage sites, implement these advanced methodological approaches using SIRT1 Antibody, Biotin Conjugated:

Laser Microirradiation with Live-Cell Imaging:

• Transfect cells with a photosensitizer (e.g., Hoechst 33342).

• Pre-label SIRT1 using a cell-permeable streptavidin conjugated to a fluorophore that binds to the biotin-conjugated SIRT1 antibody.

• Perform laser microirradiation to create localized DNA damage.

• Capture time-lapse images (intervals of 5-10 seconds for up to 10 minutes).

• Quantify fluorescence intensity at damage sites over time to measure recruitment kinetics.

Research has demonstrated that SIRT1 is rapidly mobilized to DSBs upon DNA damage, making this technique valuable for studying its real-time dynamics .

Proximity Ligation Assay (PLA):

• Induce DNA damage in cells (e.g., using ionizing radiation or radiomimetic drugs).

• Fix cells at various time points post-damage.

• Perform PLA using SIRT1 Antibody, Biotin Conjugated and antibodies against known DSB markers (γH2AX, 53BP1).

• Quantify PLA signals to assess proximity between SIRT1 and DSB markers over time.

This approach can reveal the temporal association between SIRT1 and other DNA repair factors at damage sites.

Chromatin Fractionation Combined with Proximity-dependent Biotin Identification (BioID):

• Generate a BioID-SIRT1 fusion construct.

• Induce DNA damage and allow biotin labeling of proximal proteins.

• Isolate chromatin fractions at different time points.

• Use SIRT1 Antibody, Biotin Conjugated for immunoprecipitation to compare with BioID results.

• Identify proteins that interact with SIRT1 specifically on chromatin after DNA damage.

This technique can map the changing protein interaction network of SIRT1 during the DNA damage response.

The synergistic action between SIRT1 and SIRT6 in DNA repair has been demonstrated, with SIRT1 deacetylating SIRT6 at K33, thus promoting its polymerization and recognition of DSBs. SIRT6 that is deacetylated at K33 anchors to γH2AX, allowing retention on the chromatin flanking the DSBs and subsequent chromatin remodeling via deacetylating H3K9ac and H3K56ac .

How can I resolve non-specific binding issues when using SIRT1 Antibody, Biotin Conjugated?

When encountering non-specific binding with SIRT1 Antibody, Biotin Conjugated, implement these methodological solutions:

For Western Blot Applications:

• Increase blocking stringency: Use 5% BSA instead of milk, or add 0.2% Tween-20 to the blocking buffer.

• Optimize primary antibody dilution: Test serial dilutions (1:1000, 1:2000, 1:4000, 1:6000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Reduce endogenous biotin interference: Pre-block with free avidin (10 μg/ml) for 30 minutes before adding the biotin-conjugated antibody.

• Add competitive blocking agents: Include 0.1-0.5% normal serum from the same species as the sample to reduce non-specific binding.

• Increase washing stringency: Perform 5-6 washes with TBST containing 0.1% SDS to remove loosely bound antibodies.

For Immunohistochemistry Applications:

• Quench endogenous biotin: Treat sections with avidin-biotin blocking kit before antibody incubation.

• Optimize antigen retrieval: Compare citrate buffer (pH 6.0) versus TE buffer (pH 9.0) to determine which provides optimal specific staining with minimal background.

• Apply protein blocking steps: Use 10% normal serum plus 1% BSA for 1 hour at room temperature.

• Titrate antibody concentration: Test dilutions from 1:500 to 1:2000 to identify optimal working concentration.

• Increase washing duration: Perform 3-5 washes of 10 minutes each instead of shorter washes.

For Immunoprecipitation:

• Pre-clear lysates: Incubate lysates with Protein A/G beads for 1 hour before adding the antibody.

• Use mild detergents: Include 0.1% NP-40 or Triton X-100 in wash buffers to reduce non-specific protein binding.

• Add carrier proteins: Include 0.1-0.5 mg/ml BSA in binding and wash buffers.

• Optimize salt concentration: Test wash buffers with increasing NaCl concentrations (150 mM to 300 mM) to find the optimal stringency .

What controls should be included when studying SIRT1-SIRT6 interactions using this antibody?

When investigating SIRT1-SIRT6 interactions using SIRT1 Antibody, Biotin Conjugated, include these essential controls to ensure experimental validity:

Positive Controls:

• Known SIRT1-interacting protein: Include immunoprecipitation for a well-established SIRT1 binding partner (e.g., p53) to confirm the immunoprecipitation efficiency.

• SIRT1 overexpression: Use lysates from cells overexpressing tagged SIRT1 to verify antibody specificity.

• DNA damage-induced sample: Include samples from cells treated with DNA damaging agents (e.g., etoposide) where SIRT1-SIRT6 interaction is expected to increase.

Negative Controls:

• Isotype control: Use biotin-conjugated rabbit IgG at the same concentration as the SIRT1 antibody.

• SIRT1-knockout/knockdown samples: Include lysates from SIRT1 KO/KD cells to confirm antibody specificity.

• Competitive peptide blocking: Pre-incubate antibody with the immunizing peptide to verify binding specificity.

Experimental Validation Controls:

• Reciprocal Co-IP: Perform parallel immunoprecipitation with SIRT6 antibody and blot for SIRT1 to confirm interaction from both directions.

• SIRT1 mutant expression: Include cells expressing SIRT1 catalytic mutants (H363Y) to determine if deacetylase activity affects SIRT6 interaction.

• SIRT6 K33 mutants: Include cells expressing SIRT6 K33R (deacetylation mimic) and K33Q (acetylation mimic) to verify the importance of this residue for interaction.

Sample Preparation Controls:

• Input sample: Load 5-10% of pre-immunoprecipitation lysate to verify protein expression levels.

• Supernatant sample: Analyze the post-immunoprecipitation supernatant to assess depletion efficiency.

• Beads-only control: Incubate lysate with streptavidin beads without antibody to identify non-specific binding to the beads.

Research has demonstrated that SIRT1 interacts with SIRT6 through the N-terminus of SIRT6, and that SIRT1 deacetylates SIRT6 at K33. Both SIRT1 WT and SIRT6 K33R (but not K33Q or the catalytically inactive H133Y mutant) can rescue DNA repair defects in SIRT1-deficient cells, highlighting the importance of proper controls when studying these interactions .

How can I optimize dual immunofluorescence protocols for co-localization studies of SIRT1 and SIRT6?

To optimize dual immunofluorescence protocols for co-localization studies of SIRT1 and SIRT6, employ these methodological approaches:

Sequential Double Immunostaining Protocol:

• Cell/Tissue Preparation:

  • Fix samples with 4% paraformaldehyde for 15 minutes at room temperature.

  • Permeabilize with 0.2% Triton X-100 for 10 minutes.

  • Block with 5% normal goat serum plus 1% BSA for 1 hour.

• First Primary Antibody Incubation:

  • Apply SIRT1 Antibody, Biotin Conjugated at 1:200-1:400 dilution.

  • Incubate overnight at 4°C in a humidified chamber.

  • Wash 3 times with PBS, 5 minutes each.

• First Detection Step:

  • Incubate with streptavidin conjugated to a far-red fluorophore (e.g., Alexa Fluor 647) at 1:500 for 1 hour at room temperature.

  • Wash 3 times with PBS, 5 minutes each.

• Second Primary Antibody Incubation:

  • Apply anti-SIRT6 antibody (from different host species, e.g., mouse) at manufacturer's recommended dilution.

  • Incubate overnight at 4°C or 2 hours at room temperature.

  • Wash 3 times with PBS, 5 minutes each.

• Second Detection Step:

  • Incubate with species-specific secondary antibody conjugated to a spectrally distinct fluorophore (e.g., Alexa Fluor 488) at 1:500 for 1 hour.

  • Wash 3 times with PBS, 5 minutes each.

• Nuclear Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes.

  • Mount with anti-fade mounting medium.

Critical Optimization Parameters:

• Signal Amplification for Low-Abundance Proteins:

  • For weak SIRT1 signals: Use tyramide signal amplification (TSA) system after streptavidin-HRP incubation.

  • For weak SIRT6 signals: Use multi-layer detection with biotinylated secondary antibody followed by fluorescent streptavidin.

• Background Reduction Strategies:

  • Pre-adsorb secondary reagents with cell/tissue lysates from the species being examined.

  • Include 0.1% Tween-20 in all antibody dilution buffers.

  • Perform additional blocking with 10 mg/ml BSA if non-specific binding persists.

• Co-localization Analysis Parameters:

  • Capture high-resolution z-stack images (0.3-0.5 μm steps).

  • Perform deconvolution to improve signal-to-noise ratio.

  • Quantify co-localization using Pearson's correlation coefficient and Manders' overlap coefficient.

  • Compare co-localization in normal conditions versus after DNA damage induction.

Research has shown co-localization between SIRT6 and SIRT1 by confocal microscopy in cells co-transfected with GFP-SIRT6 and DsRed-SIRT1, which increases following DNA damage. This co-localization facilitates SIRT1-mediated deacetylation of SIRT6 at K33, promoting its mobilization to DNA double-strand breaks .

How can SIRT1 Antibody, Biotin Conjugated be utilized in DNA repair pathway analyses?

SIRT1 Antibody, Biotin Conjugated can be strategically employed in multiple advanced methodologies to analyze DNA repair pathways:

Chromatin Immunoprecipitation Sequencing (ChIP-seq):

• Induce DNA damage using ionizing radiation (2-10 Gy) or radiomimetic drugs.

• Perform ChIP using SIRT1 Antibody, Biotin Conjugated coupled to streptavidin beads.

• Prepare ChIP-seq libraries and perform next-generation sequencing.

• Analyze genome-wide distribution of SIRT1 before and after DNA damage.

• Identify specific genomic regions where SIRT1 is recruited following damage.

This approach can reveal previously unknown roles of SIRT1 in recognizing specific DNA damage contexts or genomic features.

Proximity-dependent Labeling Combined with Proteomics:

• Generate cells expressing SIRT1 fused to a proximity-dependent biotin ligase (BioID or TurboID).

• Induce DNA damage and allow proximity labeling to occur.

• Isolate biotinylated proteins using streptavidin pulldown.

• Compare results with conventional immunoprecipitation using SIRT1 Antibody, Biotin Conjugated.

• Identify the dynamic interactome of SIRT1 during DNA repair.

DNA Repair Pathway-Specific Reporter Assays:

• Utilize cells with integrated reporter constructs for specific repair pathways (HR, NHEJ, SSA, etc.).

• Manipulate SIRT1 expression (overexpression, knockdown) or activity (inhibitors).

• Use SIRT1 Antibody, Biotin Conjugated to confirm expression changes or to immunoprecipitate SIRT1 complexes.

• Correlate SIRT1 status with repair pathway efficiency.

Research has shown that SIRT1 and SIRT6 work synergistically in DNA repair pathways. When SIRT1 deacetylates SIRT6 at K33, it promotes SIRT6 polymerization and recognition of DNA double-strand breaks. Subsequently, SIRT6 anchors to γH2AX, which allows it to be retained on chromatin flanking the DSBs and perform necessary chromatin remodeling through deacetylation of H3K9ac and H3K56ac .

What are the considerations for using SIRT1 Antibody, Biotin Conjugated in single-cell analysis techniques?

When adapting SIRT1 Antibody, Biotin Conjugated for single-cell analysis techniques, consider these methodological aspects:

Single-Cell Immunofluorescence Analysis:

• Optimized Fixation Protocol:

  • Test multiple fixation methods (4% PFA, methanol, or combination) to preserve epitope accessibility while maintaining cellular architecture.

  • Optimize fixation time (10-20 minutes) to prevent overfixation that might mask epitopes.

• Signal Amplification Strategies:

  • Employ tyramide signal amplification for detecting low abundance SIRT1.

  • Use quantum dots coupled to streptavidin for improved photostability during extended imaging.

  • Consider rolling circle amplification for ultra-sensitive detection in single cells.

• Multiplexing Considerations:

  • Use spectral unmixing to separate SIRT1 signal from other markers when performing multi-parameter analysis.

  • Implement sequential antibody labeling and stripping for highly multiplexed imaging.

  • Ensure compensation controls when combining with fluorophores that have spectral overlap.

Mass Cytometry (CyTOF) Applications:

• Metal Conjugation Strategy:

  • Utilize streptavidin conjugated to rare earth metals for detection of biotin-conjugated SIRT1 antibody.

  • Test multiple metal tags to identify optimal signal-to-noise ratio.

• Staining Protocol Optimization:

  • Extend permeabilization time (30-45 minutes) to ensure antibody access to nuclear SIRT1.

  • Increase antibody concentration (2-5× typical flow cytometry concentration) to compensate for reduced sensitivity.

  • Include cell barcoding to minimize batch effects across samples.

Single-Cell Western Blot Considerations:

• Protein Capture Strategy:

  • Optimize lysis conditions to ensure complete extraction of both nuclear and cytoplasmic SIRT1 pools.

  • Test multiple polyacrylamide gel densities (7-10%) to identify optimal separation conditions.

• Detection Approach:

  • Apply streptavidin-HRP at higher concentration (1:1000) than conventional Western blots.

  • Use chemiluminescent substrate with extended signal duration for reliable quantification.

  • Include on-chip normalization controls (e.g., total protein staining) for accurate relative quantification.

Research has demonstrated that SIRT1 exhibits heterogeneous expression and localization patterns across different cell types and in response to various cellular stresses, making single-cell analysis particularly valuable for understanding its context-dependent functions .

How can I apply SIRT1 Antibody, Biotin Conjugated to study the role of SIRT1 in chromatin remodeling complexes?

To investigate SIRT1's role in chromatin remodeling complexes using SIRT1 Antibody, Biotin Conjugated, implement these advanced methodological strategies:

Sequential Chromatin Immunoprecipitation (Re-ChIP):

• Perform first ChIP using antibodies against chromatin remodeling complex components (BRG1, SNF2H, etc.).

• Elute the protein-DNA complexes under mild conditions.

• Perform second ChIP using SIRT1 Antibody, Biotin Conjugated.

• Analyze co-occupancy at specific genomic loci by qPCR or sequencing.

This approach can identify genomic regions where SIRT1 cooperates with specific chromatin remodeling complexes. Research has indicated potential cooperation between SIRT1, BRG1, and PAR at DNA double-strand break sites to promote homologous recombination efficiency .

Nucleosome Remodeling Assays:

• Reconstitute nucleosomes in vitro with recombinant or purified histones.

• Immunoprecipitate SIRT1 complexes using SIRT1 Antibody, Biotin Conjugated.

• Add the immunoprecipitated complexes to the nucleosome substrates.

• Assess nucleosome sliding, eviction, or histone modification changes.

• Compare results with and without specific chromatin remodeling complex inhibitors.

High-Resolution Chromatin Structure Analysis:

• Perform Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) in cells with manipulated SIRT1 levels.

• Correlate changes in chromatin accessibility with SIRT1 binding sites identified by ChIP-seq using SIRT1 Antibody, Biotin Conjugated.

• Integrate with histone modification data (H3K9ac, H3K56ac) to build a comprehensive model of SIRT1-mediated chromatin changes.

Studies have shown that SIRT1 and SIRT6 function cooperatively in chromatin remodeling during DNA damage response. SIRT1 deacetylates SIRT6 at K33, promoting its polymerization and recognition of DSBs. This deacetylated SIRT6 then anchors to γH2AX, allowing retention on chromatin flanking DSBs where it performs chromatin remodeling through deacetylation of H3K9ac and H3K56ac .

Proximity Ligation Assay for Protein Complexes:

• Fix cells at different cell cycle stages or following DNA damage.

• Perform PLA using SIRT1 Antibody, Biotin Conjugated and antibodies against chromatin remodelers.

• Quantify and map interactions in different nuclear regions or at DNA damage sites.

• Correlate with functional outcomes using reporter assays for transcription or DNA repair.

This technique can reveal spatial and temporal dynamics of SIRT1's associations with chromatin remodeling machinery in response to cellular signals or stresses.

How should I interpret differences in SIRT1 localization data between techniques when using biotin-conjugated antibodies?

When interpreting differences in SIRT1 localization data across techniques using SIRT1 Antibody, Biotin Conjugated, consider these methodological insights:

Common Discrepancies Between Techniques:

Methodological Considerations for Accurate Interpretation:

• Fixation-Dependent Artifacts:

  • Paraformaldehyde fixation may preserve cytoplasmic SIRT1 better than methanol fixation.

  • Compare results using 1-4% paraformaldehyde with varying fixation times (10-30 minutes).

  • Always include parallel samples with different fixation protocols when establishing localization patterns.

• Antibody Accessibility Issues:

  • Biotin conjugation may alter antibody penetration into dense nuclear regions.

  • Compare direct detection using biotinylated antibody versus indirect detection using unconjugated primary and biotinylated secondary antibodies.

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100 for 5-15 minutes) to ensure nuclear access.

• Resolution Limitations:

  • Standard fluorescence microscopy may not resolve fine subnuclear structures.

  • Employ super-resolution techniques (STORM, STED) for detailed localization studies.

  • Use Z-stack acquisition with deconvolution to improve discrimination between nuclear and perinuclear signals.

Research has demonstrated that SIRT1 exhibits dynamic localization, redistributing on chromatin upon DNA damage to deacetylate repair factors such as XPA, NBS1, and Ku70. In addition, confocal microscopy has revealed co-localization between SIRT6 and SIRT1 in cell nuclei, which is functionally significant for their synergistic roles in DNA damage response .

What are the best practices for quantifying SIRT1-SIRT6 interactions in different experimental systems?

When quantifying SIRT1-SIRT6 interactions across experimental systems, implement these best practices:

Co-Immunoprecipitation Quantification:

• Standardized Normalization Approach:

  • Always normalize co-IP signal to both input levels and IP efficiency.

  • Calculate interaction ratio: (SIRT6 in SIRT1-IP / SIRT1 in IP) / (SIRT6 in input / SIRT1 in input).

  • Include serial dilutions of input samples to ensure detection is in the linear range.

• Statistical Analysis:

  • Perform minimum of three biological replicates for statistical validity.

  • Use paired statistical tests when comparing treatments within the same experimental batch.

  • Report both fold changes and p-values when describing interaction differences.

Fluorescence Resonance Energy Transfer (FRET) Analysis:

• Control Measurements:

  • Include donor-only and acceptor-only controls for spectral bleedthrough correction.

  • Use non-interacting protein pairs with similar localization as negative controls.

  • Use fusion proteins with fixed distances between fluorophores as positive controls.

• Quantification Methods:

  • Calculate FRET efficiency using acceptor photobleaching or sensitized emission.

  • Report FRET values as distance estimations (nm) when possible.

  • Analyze FRET signals in different subcellular compartments separately.

Proximity Ligation Assay Quantification:

• Signal Analysis Parameters:

  • Count discrete PLA spots per nucleus.

  • Measure average spot intensity and size as indicators of interaction strength.

  • Analyze spatial distribution of spots relative to nuclear regions or damage sites.

• Normalization Strategy:

  • Normalize to cell or nuclear area when comparing different cell types.

  • Include SIRT1 and SIRT6 single antibody controls to establish background thresholds.

  • Validate with SIRT1 or SIRT6 knockdown controls.

Research has shown that SIRT1 interacts with SIRT6 through the N-terminus of SIRT6 and deacetylates it at K33. This interaction and subsequent deacetylation promote SIRT6 polymerization and recognition of DNA double-strand breaks, facilitating DNA repair .

When interpreting interaction data, consider that SIRT1-SIRT6 interactions may be enhanced following DNA damage and that specific mutations (e.g., SIRT6 K33R, K33Q) can affect this interaction. The K33ac-containing peptide has been demonstrated to effectively block the in vitro binding of SIRT6 to SIRT1, confirming the specificity of this interaction .

What emerging methodologies could enhance the utility of SIRT1 Antibody, Biotin Conjugated in epigenetic research?

Several cutting-edge methodologies hold promise for expanding the utility of SIRT1 Antibody, Biotin Conjugated in epigenetic research:

CUT&RUN (Cleavage Under Targets and Release Using Nuclease):

• Immobilize intact cells on concanavalin A-coated magnetic beads.

• Add SIRT1 Antibody, Biotin Conjugated followed by streptavidin-conjugated protein A-MNase.

• Activate targeted chromatin digestion with calcium.

• Isolate released DNA fragments for sequencing.

• Achieve higher signal-to-noise ratio than conventional ChIP-seq.

This approach would provide higher resolution mapping of SIRT1 genomic occupancy with less background and fewer cells than traditional ChIP-seq, enabling more precise correlation between SIRT1 binding and specific epigenetic states.

Live-Cell Epigenetic Tracking:

• Generate cell lines expressing minimal epitope-tagged SIRT1.

• Use cell-permeable biotin-conjugated anti-epitope Fab fragments.

• Add streptavidin-conjugated quantum dots for long-term tracking.

• Perform live-cell imaging to track SIRT1 dynamics during epigenetic transitions.

This method could reveal the real-time kinetics of SIRT1 recruitment to specific chromatin regions during cellular responses to various stimuli or developmental transitions.

Single-Cell Multi-Omics Integration:

• Perform single-cell ATAC-seq or single-cell ChIP-seq for SIRT1 using the biotin-conjugated antibody.

• In parallel, analyze transcriptome (scRNA-seq) from the same cells.

• Integrate datasets to correlate SIRT1 binding patterns with gene expression changes at single-cell resolution.

This approach could uncover previously unappreciated heterogeneity in SIRT1's epigenetic functions across cell populations and identify cell state-specific roles.

CRISPR-Based Genomic Recruitment:

• Fuse catalytically inactive Cas9 (dCas9) with a biotin acceptor peptide.

• Use guide RNAs to target dCas9 to specific genomic loci.

• Add SIRT1 Antibody, Biotin Conjugated and streptavidin to create a bridge.

• Artificially recruit endogenous SIRT1 to desired genomic sites.

• Analyze resulting epigenetic and transcriptional changes.

This synthetic approach could help dissect the direct epigenetic consequences of SIRT1 recruitment to specific genomic contexts.

Research has demonstrated that SIRT1 plays critical roles in chromatin regulation, particularly in the context of DNA damage where it works synergistically with SIRT6 to facilitate chromatin remodeling and DNA repair .

How might SIRT1 Antibody, Biotin Conjugated be used to investigate the crosstalk between DNA repair and epigenetic regulation?

SIRT1 Antibody, Biotin Conjugated offers unique opportunities to explore the intersection between DNA repair and epigenetic regulation through these advanced methodological approaches:

Sequential ChIP-seq for Repair and Epigenetic Factors:

• Perform first ChIP using antibodies against DNA repair proteins (γH2AX, 53BP1, BRCA1).

• Elute complexes and perform second ChIP with SIRT1 Antibody, Biotin Conjugated.

• Sequence and map genomic regions where SIRT1 co-localizes with repair machinery.

• Correlate these regions with specific chromatin states or genomic features.

This approach would identify specific chromatin contexts where SIRT1 participates in DNA repair processes.

Epigenetic Dynamics at DNA Damage Sites:

• Induce site-specific DNA damage using CRISPR-Cas9 or laser microirradiation.

• Perform ChIP using SIRT1 Antibody, Biotin Conjugated at various time points post-damage.

• In parallel, analyze histone modification changes (H3K9ac, H3K56ac, γH2AX).

• Construct a temporal map of SIRT1 recruitment and epigenetic changes during repair.

Research has demonstrated that SIRT1 and SIRT6 work synergistically in DNA repair, with SIRT1 deacetylating SIRT6 at K33, promoting its polymerization and recognition of DNA double-strand breaks. SIRT6, in turn, anchors to γH2AX and performs chromatin remodeling through deacetylation of H3K9ac and H3K56ac .

Nascent Chromatin Capture with SIRT1 Profiling:

• Label newly synthesized DNA with EdU during repair synthesis.

• Isolate EdU-containing chromatin fragments.

• Perform ChIP on these fragments using SIRT1 Antibody, Biotin Conjugated.

• Compare SIRT1 occupancy on nascent versus mature chromatin during repair.

This technique could reveal SIRT1's role in establishing or maintaining epigenetic patterns on newly synthesized DNA during repair processes.

Targeted Proteomics of SIRT1 Complexes:

• Immunoprecipitate SIRT1 using SIRT1 Antibody, Biotin Conjugated before and after DNA damage.

• Perform quantitative mass spectrometry to identify damage-specific interaction partners.

• Use targeted proteomics to quantify specific post-translational modifications on histones and repair factors associated with SIRT1.

• Construct interaction networks that change during the damage response.

This approach would identify the dynamic composition of SIRT1-containing complexes during DNA repair, providing insights into how SIRT1 coordinates repair and epigenetic functions.