SIRT1 Antibody, FITC conjugated

What is SIRT1 and why is it significant in cellular research?

SIRT1 (Silent Mating Type Information Regulation 2 Homolog 1) is a NAD-dependent protein deacetylase that plays crucial roles in maintaining genome stability by preventing the activation of latent replication origins. It is also known by several alternative names including SIR2-like protein 1, SIR2alpha, and SIR2L1 . SIRT1 has significant implications in research related to apoptosis, chromatin research, cognition and behavior, DNA repair, epigenetics, and histone deacetylation . Recent studies indicate that SIRT1 may stabilize extrachromosomal amplicons and facilitate gene amplification, which has important implications in cancer malignancy and protein expression . The protein is localized in both the cytoplasm and nucleus, making it an interesting target for subcellular localization studies using fluorescently labeled antibodies .

What are the key differences between monoclonal and polyclonal SIRT1-FITC antibodies?

Monoclonal SIRT1-FITC antibodies, such as the Mouse Monoclonal 1F3 (NBP151641F) and Rabbit Monoclonal 012 (NBP2-89977F), are derived from a single B-cell clone and recognize a specific epitope within SIRT1 . These antibodies offer high specificity and consistency between batches, making them ideal for quantitative analyses. The monoclonal antibodies in the search results target specific regions of SIRT1 - for example, the 1F3 clone targets amino acids 265-452 of human SIRT1 .

In contrast, polyclonal SIRT1-FITC antibodies, like those from rabbit hosts, recognize multiple epitopes on the SIRT1 protein . The polyclonal antibody described in search result was raised against a synthetic peptide corresponding to amino acids 580-630 of human SIRT1 . Polyclonal antibodies typically provide higher sensitivity due to their ability to bind multiple epitopes but may have more batch-to-batch variation. The choice between monoclonal and polyclonal depends on experimental goals - monoclonals for high specificity requirements and polyclonals for applications requiring stronger signal detection across varied conditions.

How does FITC conjugation affect experimental design with SIRT1 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation to SIRT1 antibodies creates a direct detection system that eliminates the need for secondary antibodies in fluorescence-based applications. The FITC-conjugated SIRT1 antibodies have specific excitation and emission profiles (typically excitation at 494-495nm and emission at 518-519nm) . This spectral characteristic requires careful consideration when designing multi-color experiments to avoid fluorescence overlap with other fluorophores.

When working with FITC-conjugated antibodies, researchers must account for several experimental factors: 1) FITC is susceptible to photobleaching, necessitating protection from light during storage and handling; 2) The fluorescence intensity of FITC is pH-dependent, with optimal performance at slightly alkaline conditions; 3) Sample autofluorescence in the green spectrum may interfere with FITC detection, requiring appropriate controls and background subtraction strategies . Additionally, the conjugation process might theoretically affect antibody binding affinity in some cases, so validation in each experimental system is recommended.

What applications are supported by commercially available SIRT1-FITC antibodies?

Available SIRT1-FITC antibodies support multiple research applications with varying degrees of validation. The following table summarizes the applications validated for different SIRT1-FITC antibody products:

For flow cytometry applications, the Mouse Monoclonal SIRT1-FITC antibody is particularly useful for analyzing SIRT1 expression at the single-cell level . Immunofluorescence and immunocytochemistry applications benefit from both monoclonal and polyclonal options, with the polyclonal antibody C04019F specifically optimized for immunofluorescence studies . For ELISA-based quantitative analysis of SIRT1, both the mouse monoclonal (NBP151641F) and rabbit monoclonal (NBP2-89977F) antibodies have been validated .

What are the recommended storage conditions for preserving SIRT1-FITC antibody activity?

Proper storage is critical for maintaining the activity of FITC-conjugated SIRT1 antibodies. The primary recommendations across products include:

• Temperature: Store at 4°C for short-term use (up to 12 months) . For long-term storage, some products recommend -20°C .

• Light protection: All FITC-conjugated antibodies must be stored in the dark to prevent photobleaching of the fluorophore .

• Buffer conditions: Products are typically formulated in stabilizing buffers such as PBS with 0.05% sodium azide or TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% glycerol .

• Freeze-thaw cycles: Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody and the FITC conjugate .

For optimal performance, it's recommended to aliquot the antibody upon receipt to minimize freeze-thaw cycles if stored at -20°C. When removing from storage, allow the antibody to equilibrate to room temperature before opening to prevent condensation that could introduce contamination or affect concentration .

What controls should be included when using SIRT1-FITC antibodies in experimental procedures?

When designing experiments with SIRT1-FITC antibodies, several essential controls should be included:

• Isotype control: An irrelevant antibody of the same isotype (IgG1 for mouse monoclonal or IgG for rabbit antibodies ) conjugated to FITC. This controls for non-specific binding due to Fc receptor interactions or other isotype-specific binding.

• Negative tissue/cell controls: Samples known to lack SIRT1 expression or SIRT1-knockout cell lines should be used to establish background fluorescence levels and verify antibody specificity .

• Blocking controls: Pre-incubation of the antibody with the immunizing peptide (where available) to demonstrate binding specificity. For recombinant-derived antibodies, the relevant recombinant fragments can be used (e.g., amino acids 265-452 for NBP151641F or Met 193-Ser 747 for NBP2-89977F ).

• Subcellular localization validation: Since SIRT1 localizes to both cytoplasm and nucleus , appropriate subcellular markers should be included to verify the expected localization pattern.

• Autofluorescence control: Unstained samples to account for natural fluorescence in the FITC emission spectrum, particularly important in tissues with high autofluorescence like liver or kidney.

These controls help distinguish true signal from technical artifacts and validate experimental findings with SIRT1-FITC antibodies.

How can SIRT1-FITC antibodies be utilized in studies of extrachromosomal gene amplification?

SIRT1-FITC antibodies offer valuable tools for investigating SIRT1's role in extrachromosomal gene amplification. Recent research indicates that SIRT1 maintains genome stability by preventing the activation of latent replication origins and may be involved in stabilizing extrachromosomal amplicons . To study this phenomenon, researchers can employ several approaches:

• Co-localization studies: SIRT1-FITC antibodies can be used alongside DNA markers to visualize the spatial relationship between SIRT1 and extrachromosomal DNA structures like double minutes (DMs). This requires high-resolution imaging techniques like confocal microscopy.

• SIRT1 knockout comparison: Using SIRT1-FITC antibodies to compare extrachromosomal DNA content between wild-type and SIRT1-knockout cells (like the COLO 320DM model described in the research) can help quantify SIRT1's impact on extrachromosomal amplification .

• Time-course experiments: SIRT1-FITC antibodies can track changes in SIRT1 localization during the formation of extrachromosomal elements, potentially revealing temporal relationships between SIRT1 activity and amplicon stability.

• Flow cytometric analysis: The flow cytometry-validated SIRT1-FITC antibodies can be used to sort cells based on SIRT1 expression levels, followed by analysis of extrachromosomal DNA content in SIRT1-high versus SIRT1-low populations.

These approaches leverage the fluorescent properties of FITC-conjugated antibodies to investigate the mechanistic relationship between SIRT1 and the formation or maintenance of extrachromosomal gene amplifications.

What considerations are important when using phospho-specific SIRT1-FITC antibodies?

The phospho-Ser47 specific SIRT1-FITC antibody enables researchers to study post-translational regulation of SIRT1 function. When working with phospho-specific antibodies, several special considerations apply:

• Sample preparation: Phosphorylation status is highly sensitive to sample handling. Phosphatase inhibitors must be included in all buffers during sample preparation to prevent dephosphorylation.

• Validation strategy: Experimental validation should include treatment with phosphatases to demonstrate specificity for the phosphorylated form, and treatment with kinase activators/inhibitors known to affect SIRT1 Ser47 phosphorylation.

• Comparative analysis: Parallel staining with both phospho-specific and total SIRT1 antibodies (potentially with different fluorophores) allows calculation of the phosphorylated fraction of SIRT1, providing insights into its activation state.

• Stimulus response: Phospho-Ser47 SIRT1 levels may change rapidly in response to cellular signaling events, requiring careful timing of fixation after experimental treatments.

• Quantification methods: When quantifying phospho-SIRT1 signals, normalization to total SIRT1 is preferable to housekeeping proteins to account for variations in SIRT1 expression levels between samples.

These methodological considerations ensure that experiments with phospho-specific SIRT1-FITC antibodies yield meaningful data about the regulation of SIRT1 activity through phosphorylation mechanisms.

What are common causes of background fluorescence when using SIRT1-FITC antibodies?

Background fluorescence can significantly impact the signal-to-noise ratio when using SIRT1-FITC antibodies. Common causes and their potential solutions include:

• Sample autofluorescence: Biological samples, particularly those containing lipofuscin, NADH, flavins, or collagen, naturally fluoresce in the FITC emission range. This can be mitigated by:

  • Using longer wavelength fluorophores for tissues with high autofluorescence

  • Treating samples with Sudan Black B or commercial autofluorescence quenching reagents

  • Implementing spectral unmixing during image acquisition

• Non-specific antibody binding: This may occur due to:

  • Fc receptor interactions: Block with appropriate serum or commercial Fc receptor blocking reagents

  • Hydrophobic interactions: Increase BSA or serum concentration in blocking and antibody diluent buffers

  • Insufficient blocking: Extend blocking time and optimize blocker concentration

• Fixation artifacts: Overfixation with aldehyde-based fixatives can create autofluorescent byproducts and expose epitopes leading to non-specific binding. Optimize fixation protocols by:

  • Reducing fixation time or concentration

  • Implementing antigen retrieval methods

  • Testing alternative fixatives appropriate for SIRT1 detection

• Buffer composition: The presence of certain compounds in buffers can contribute to background. Ensure buffers are freshly prepared and free of contaminants. The appropriate buffer compositions for SIRT1-FITC antibodies include PBS with 0.05% sodium azide or TBS (pH 7.4) with specific stabilizers .

Implementing these strategies can significantly improve signal specificity when working with SIRT1-FITC antibodies in fluorescence-based applications.

How can researchers address inconsistent staining patterns with SIRT1-FITC antibodies?

Inconsistent staining patterns with SIRT1-FITC antibodies can arise from several sources. Systematic troubleshooting approaches include:

• Antibody titration: Determine the optimal concentration through careful titration experiments. Starting recommendations vary by antibody and application, but researchers should experimentally determine the optimal dilution for their specific samples .

• Sample preparation consistency:

  • Standardize fixation protocols (time, temperature, fixative concentration)

  • Ensure consistent cell/tissue permeabilization

  • Optimize antigen retrieval methods if necessary

  • Maintain consistent incubation times and temperatures

• Antibody validation across lots:

  • Compare new antibody lots against previously validated lots

  • Maintain positive control samples that work well with existing lots

  • Consider creating a standard curve with recombinant SIRT1 protein

• SIRT1 biology considerations:

  • Expression levels vary across cell types and conditions

  • Subcellular localization can shift between nucleus and cytoplasm

  • Post-translational modifications may affect epitope accessibility

  • Cell cycle variations may impact SIRT1 expression patterns

• Experimental timing: For proteins involved in stress responses like SIRT1, minor variations in experimental timing, cell confluency, or environmental stressors can affect expression and localization patterns.

By systematically addressing these factors, researchers can achieve more consistent staining patterns with SIRT1-FITC antibodies across experiments.

How should researchers interpret differences in SIRT1 localization between cytoplasmic and nuclear compartments?

SIRT1 is known to localize to both the cytoplasm and nucleus , with its distribution potentially providing important functional insights. When interpreting SIRT1-FITC staining patterns:

• Normal distribution pattern: In most cells, SIRT1 shows both nuclear and cytoplasmic localization, with the balance varying by cell type, cell cycle stage, and physiological conditions.

• Shuttling mechanisms: SIRT1 contains both nuclear localization signals (NLS) and nuclear export signals (NES). Changes in the relative strength of these signals through post-translational modifications can alter subcellular distribution.

• Functional implications:

  • Nuclear SIRT1 typically associates with chromatin remodeling, transcriptional regulation, and DNA repair functions

  • Cytoplasmic SIRT1 often relates to cytoskeletal regulation, mitochondrial functions, and cytoplasmic protein deacetylation

  • Changes in the nuclear/cytoplasmic ratio may indicate cellular stress responses or altered signaling pathways

• Quantification approaches: When quantifying localization patterns, researchers should:

  • Use nuclear and cytoplasmic markers for accurate compartment identification

  • Calculate nuclear/cytoplasmic ratios rather than absolute intensities

  • Compare patterns across multiple cells to account for biological variability

  • Consider cell cycle stage, which may influence SIRT1 distribution

• Technical considerations: Fixation methods can affect apparent localization of SIRT1. Cross-validation with different fixation protocols and antibody clones targeting different epitopes can provide more reliable localization data.

Understanding these aspects of SIRT1 biology helps researchers correctly interpret localization patterns observed with FITC-conjugated SIRT1 antibodies in their experimental systems.

What are key considerations when designing flow cytometry experiments with SIRT1-FITC antibodies?

Flow cytometry with SIRT1-FITC antibodies requires careful experimental design. Key considerations include:

• Fluorophore compatibility: FITC (excitation ~494-495nm, emission ~518-519nm) occupies the standard green fluorescence channel (FL1 on many cytometers). When designing multi-color panels:

  • Avoid fluorophores with substantial spectral overlap (PE, GFP)

  • Include single-color controls for compensation

  • Consider alternative conjugates for multi-parameter analysis

• Sample preparation protocol:

  • Fixation: Most protocols require formaldehyde fixation (2-4%)

  • Permeabilization: Since SIRT1 is intracellular, effective permeabilization with detergents (0.1% Triton X-100 or saponin-based buffers) is essential

  • Cell concentration: Typically 1×10^6 cells/mL for optimal detection

• Controls specific for SIRT1 analysis:

  • Unstained cells for autofluorescence assessment

  • Isotype-FITC control matching the SIRT1 antibody host and isotype (IgG1 for mouse monoclonal or IgG for rabbit antibodies )

  • Positive controls: Cell lines known to express high SIRT1 levels

  • Negative controls: SIRT1 knockout or knockdown cells if available

• Analysis considerations:

  • Gating strategy should account for cell cycle phases, as SIRT1 expression may vary

  • Consider median fluorescence intensity rather than percent positive for more quantitative comparisons

  • For phospho-SIRT1 , include treatment conditions known to modulate phosphorylation status

By addressing these considerations, researchers can develop robust flow cytometry protocols for analyzing SIRT1 expression and regulation at the single-cell level.

How can researchers validate the specificity of their SIRT1-FITC antibody results?

Validating SIRT1-FITC antibody specificity is crucial for reliable research outcomes. A comprehensive validation strategy includes:

• Genetic validation approaches:

  • SIRT1 knockout models: Compare staining between wild-type and SIRT1 knockout cells

  • siRNA/shRNA knockdown: Demonstrate reduced signal with SIRT1 knockdown

  • Overexpression systems: Show increased signal with SIRT1 overexpression

• Biochemical validation:

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • For recombinant-derived antibodies, use the specific recombinant fragments (e.g., amino acids 265-452 or Met 193-Ser 747 )

  • Western blot correlation: Confirm that fluorescence intensity correlates with protein levels detected by Western blot

• Cross-antibody validation:

  • Compare staining patterns between different SIRT1 antibody clones targeting distinct epitopes

  • For phospho-specific antibodies , validate with phosphatase treatment and kinase modulators

• Functional correlation:

  • Verify that changes in SIRT1-FITC signal correlate with expected biological responses

  • For example, conditions known to activate SIRT1 should show measurable changes in phospho-SIRT1 levels

• Method orthogonality:

  • Confirm findings using alternative detection methods like mass spectrometry

  • Correlate immunofluorescence results with functional assays of SIRT1 activity