SIRT2 Antibody, FITC conjugated

What is SIRT2 and what cellular functions does it regulate?

SIRT2 (Sirtuin 2) is an NAD-dependent protein deacetylase that primarily deacetylates the 'Lys-40' of alpha-tubulin and internal lysines on histones and many other proteins, including key transcription factors . It belongs to the silent information regulator (SIR2) family of genes that are highly conserved from prokaryotes to eukaryotes . SIRT2 participates in multiple biological processes including cell cycle control, genomic integrity maintenance, microtubule dynamics regulation, cell differentiation, metabolic network modulation, and autophagy . It plays a particularly critical role in controlling mitotic exit in the cell cycle through its regulation of cytoskeleton dynamics . The gene is located on chromosome 19q13.2 in humans and 7A3 in mice .

How does FITC conjugation enhance SIRT2 antibody applications?

SIRT2 Antibody, FITC conjugated combines the specificity of SIRT2 detection with the fluorescent properties of FITC (Fluorescein Isothiocyanate). The conjugation has an absorbance/excitation peak at 494nm, which enables direct visualization in fluorescence-based applications without requiring secondary antibodies . This conjugation allows for direct detection of SIRT2 in techniques such as flow cytometry, immunofluorescence microscopy, and high-content screening. The covalent attachment of FITC to the antibody provides stable fluorescent signal while maintaining the antibody's binding specificity and affinity for SIRT2 protein.

What are the recommended experimental applications for SIRT2 Antibody, FITC conjugated?

The SIRT2 Polyclonal Antibody, FITC Conjugated has been validated for Western Blot applications according to manufacturer specifications . While Western Blot is the primary confirmed application, the FITC conjugation makes this antibody potentially suitable for:

• Immunofluorescence microscopy for subcellular localization studies

• Flow cytometry for quantitative analysis of SIRT2 expression in cell populations

• Confocal microscopy for co-localization studies with other proteins

• Live-cell imaging for tracking SIRT2 dynamics in real-time

When designing experiments, researchers should conduct preliminary validation tests in their specific experimental systems before proceeding with full-scale studies.

What are the storage and handling recommendations for maintaining antibody integrity?

The SIRT2 Antibody, FITC conjugated is supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol at a concentration of 1μg/μl (100μl total volume) . For optimal performance:

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• When thawing, allow the antibody to reach room temperature slowly before use

• Protect from prolonged light exposure during experimental procedures

• Centrifuge briefly before opening the vial to collect liquid at the bottom

• Use appropriate negative controls in experiments to account for potential non-specific binding

How should SIRT2 Antibody, FITC conjugated be validated for specific research applications?

A systematic validation approach should include:

Validation is particularly important as this antibody has been shown to recognize Mouse and Rat antigens , and cross-reactivity with human samples should be experimentally confirmed.

How can SIRT2 Antibody, FITC conjugated be used to study cell cycle regulation?

SIRT2 plays a major role in cell cycle progression and genomic stability, functioning in the antephase checkpoint that prevents precocious mitotic entry in response to microtubule stress agents . To investigate this function:

• Synchronize cells at different cell cycle stages (G1, S, G2/M) using standard methods such as double thymidine block or nocodazole treatment

• Fix cells and immunostain with SIRT2 Antibody, FITC conjugated

• Co-stain with cell cycle markers (e.g., phospho-histone H3 for mitosis)

• Analyze by flow cytometry or fluorescence microscopy to correlate SIRT2 expression/localization with cell cycle phases

• Compare SIRT2 distribution in normal versus microtubule-disrupted cells (treated with nocodazole or taxol)

This approach can reveal how SIRT2 redistributes during cell cycle progression and how it responds to conditions that activate the antephase checkpoint.

What protocols can optimize Western blot analysis using SIRT2 Antibody, FITC conjugated?

While FITC conjugation is generally optimized for fluorescence-based techniques, the following protocol modifications can enhance Western blot performance:

• Sample preparation:

  • Include protease inhibitors and phosphatase inhibitors in lysis buffer

  • Maintain cold temperatures throughout extraction

  • Use RIPA buffer for efficient extraction of nuclear and cytoplasmic proteins

• Gel electrophoresis and transfer:

  • Use 10-12% acrylamide gels for optimal resolution

  • Perform wet transfer at 30V overnight at 4°C for complete transfer

• Blocking and antibody incubation:

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

  • Incubate with SIRT2 Antibody, FITC conjugated (1:500-1:1000 dilution) overnight at 4°C

  • Protect from light during incubation

• Detection options:

  • For chemiluminescence: No secondary antibody needed if using anti-FITC HRP conjugate

  • For fluorescence: Direct visualization using a fluorescence scanner with appropriate filter (excitation ~490nm, emission ~520nm)

• Controls:

  • Include recombinant SIRT2 protein as positive control

  • Include lysate from SIRT2 knockout/knockdown cells as negative control

How can SIRT2 Antibody, FITC conjugated be used to investigate intestinal barrier integrity mechanisms?

Recent research has demonstrated that SIRT2 inhibition improves gut epithelial barrier integrity and protects against inflammatory bowel disease (IBD) . The SIRT2 Antibody, FITC conjugated can be utilized to investigate the mechanisms involved:

• Intestinal epithelial cell model:

  • Grow Caco2 cells (human colon cell line) on transwell inserts to form monolayers

  • Treat with SIRT2 inhibitors (e.g., TM, AGK2) or SIRT2 siRNA knockdown

  • Measure transepithelial electrical resistance (TEER) to assess barrier function

• ARF6 activation assessment:

  • Pull down ARF6-GTP using GST-GGA3 fusion protein

  • Perform Western blot to compare ARF6-GTP levels between control and SIRT2-inhibited conditions

  • Use SIRT2 Antibody, FITC conjugated to confirm knockdown efficiency

• E-cadherin endocytosis visualization:

  • Biotinylate cell surface proteins of intestinal epithelial cells

  • Compare surface E-cadherin levels in control versus SIRT2-inhibited conditions

  • Use immunofluorescence with SIRT2 Antibody, FITC conjugated to correlate SIRT2 localization with E-cadherin distribution

This experimental approach can help elucidate how SIRT2 regulates intestinal barrier function through the ARF6-mediated endocytosis of E-cadherin .

What explains the differential effects of SIRT2 genetic knockout versus pharmacological inhibition in disease models?

A fascinating research question emerges from the apparently contradictory reports that genetic knockout of SIRT2 aggravates IBD symptoms while pharmacological inhibition alleviates them . Researchers can investigate this phenomenon using SIRT2 Antibody, FITC conjugated through:

• Comparative activity profiling:

  • Generate SIRT2 knockout cell lines using CRISPR/Cas9

  • Treat wild-type cells with selective SIRT2 inhibitors at various concentrations

  • Use SIRT2 Antibody, FITC conjugated for immunoprecipitation followed by activity assays against different substrates

  • Compare the substrate-specific deacetylation activities between knockout and inhibitor-treated conditions

• Substrate-specific effects assessment:

  • Identify key substrates affected differently by knockout versus inhibition

  • Use SIRT2 Antibody, FITC conjugated in proximity ligation assays to visualize SIRT2-substrate interactions

  • Compare acetylation status of specific substrates (e.g., tubulin, histones) using acetyl-specific antibodies

• PROTAC versus inhibitor comparison:

  • Compare effects of SIRT2 inhibitors with PROTAC degraders of SIRT2

  • Use SIRT2 Antibody, FITC conjugated to monitor protein levels and localization

  • Assess downstream effects on ARF6 activation and E-cadherin endocytosis

This investigation supports the hypothesis that SIRT2 inhibitors block only some activities of SIRT2 rather than eliminating all functions as occurs in genetic knockout .

How can SIRT2 Antibody, FITC conjugated be used in multiparameter analysis of chromatin regulation?

SIRT2 associates with chromatin at transcriptional start sites (TSSs) and enhancers of active genes . The following experimental approach can elucidate its role in chromatin regulation:

• Chromatin immunoprecipitation (ChIP) optimization:

  • Perform ChIP using SIRT2 Antibody, FITC conjugated

  • Analyze precipitated DNA by qPCR or sequencing to identify binding sites

  • Compare SIRT2 binding profiles with histone modification maps

• Multiparameter flow cytometry:

  • Fix and permeabilize cells using optimized protocols for nuclear proteins

  • Stain with SIRT2 Antibody, FITC conjugated

  • Co-stain with antibodies against histone modifications (using different fluorophores)

  • Analyze correlation between SIRT2 levels and specific histone marks

• Super-resolution microscopy:

  • Perform STORM or PALM imaging using SIRT2 Antibody, FITC conjugated

  • Co-localize SIRT2 with transcription factors and chromatin markers

  • Analyze spatial relationships at nanometer resolution

This approach can provide insights into how SIRT2 contributes to epigenetic regulation and transcriptional control in various cellular contexts.

What are emerging applications for SIRT2 Antibody, FITC conjugated in disease research?

The SIRT2 Antibody, FITC conjugated offers significant potential for investigating SIRT2's role in various diseases:

• Inflammatory bowel disease: Building on recent findings that SIRT2 inhibition protects against IBD , researchers can use this antibody to further characterize epithelial barrier mechanisms and develop potential therapeutic approaches.

• Neurodegenerative disorders: SIRT2 has been implicated in Parkinson's and Alzheimer's diseases through its effects on protein aggregation and neuroinflammation. The fluorescently labeled antibody enables visualization of SIRT2 in neuronal cell models and brain tissue sections.

• Cancer biology: Given SIRT2's role in cell cycle regulation and genomic stability , the antibody can be used to investigate how SIRT2 expression and localization changes in different cancer types and in response to treatments.

• Metabolic diseases: SIRT2 functions in metabolic networks suggest its importance in diabetes and obesity research, where the antibody could track SIRT2 dynamics in metabolically active tissues.