SESN2 Antibody, FITC conjugated

What is SESN2 and why is it important in cellular research?

Sestrin2 (SESN2) is a highly conserved stress-inducible protein that functions as a key regulator in various cellular processes. It plays critical roles in reducing peroxiredoxins, regulating metabolic homeostasis, and protecting cells against oxidative and endoplasmic reticulum (ER) stress . SESN2 is widely expressed in tissues and localizes predominantly in the cytoplasm with occasional nuclear presence . Its expression is upregulated in response to various stressors including hypoxia, oxidative stress, and endoplasmic reticulum stress, making it an important marker for cellular stress responses in experimental research .

What applications are SESN2 antibody FITC conjugates verified for?

SESN2 antibody FITC conjugates have been verified for multiple research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunohistochemistry (IHC)

• Immunoprecipitation (IP)

• Western Blotting (WB)

Some formulations may have additional verified applications, with ELISA being the most consistently reported application across different commercial preparations .

What is the typical reagent profile of SESN2 antibody FITC conjugates?

The typical reagent profile includes:

Information compiled from product specifications

How should I optimize SESN2 antibody dilutions for different applications?

Optimal dilution ranges vary by application:

Begin with manufacturer's recommendations and optimize based on your specific experimental conditions. When studying stress-induced SESN2 expression, consider running parallel samples with and without stressors to establish baseline and induced expression levels .

What controls should be included when using SESN2 antibody in experiments?

For rigorous research with SESN2 antibody, include these controls:

• Positive Controls:

  • Cell lines with confirmed SESN2 expression (e.g., HUVECs treated with AngII show increased SESN2 expression)

  • Tissues with known SESN2 expression (widely expressed but particularly high in certain stress conditions)

• Negative Controls:

  • SESN2 knockout samples (SESN2−/− cells or tissues)

  • Isotype controls with irrelevant antibody of same class conjugated to FITC

  • Secondary antibody-only controls (for unconjugated primary antibodies)

• Validation Controls:

  • siRNA knockdown of SESN2 (to confirm antibody specificity)

  • Peptide competition assay (pre-incubating antibody with immunizing peptide)

  • SESN2 overexpression samples

Implementing these controls is crucial for result interpretation, especially when studying subtle changes in SESN2 expression under different experimental conditions .

How can I effectively use SESN2 antibody FITC conjugates for co-localization studies?

Co-localization studies with SESN2 require careful planning:

• Sample Preparation:

  • Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 (5-10 minutes)

  • Block with 5% normal serum from host species of secondary antibody

• Staining Approach:

  • Use SESN2-FITC antibody (green fluorescence) at 1:100-1:200 dilution

  • For organelle co-localization, use organelle-specific trackers or antibodies with contrasting fluorophores:

    • ER-tracker (red) for endoplasmic reticulum co-localization

    • Anti-ATF4 antibody for stress response pathway interactions

    • MitoTracker for mitochondrial association studies

• Imaging Considerations:

  • Control for bleed-through between channels

  • Use sequential scanning rather than simultaneous acquisition

  • Include single-stained controls for each fluorophore

  • Analysis should quantify Pearson's correlation coefficient or Mander's overlap coefficient

Research has demonstrated SESN2 co-localization with endoplasmic reticulum and interaction with ATF4 during stress responses, particularly in dendritic cells under stress conditions .

How can I use SESN2 antibody to investigate the role of SESN2 in cellular stress responses?

Investigating SESN2 in stress responses requires multi-dimensional experimental approaches:

• Stress Induction Protocols:

  • Oxidative stress: H₂O₂ (0.1-1mM), rotenone treatment

  • ER stress: tunicamycin (1-10μg/ml), thapsigargin (100-500nM)

  • Hypoxia: 1% O₂ conditions or CoCl₂ treatment

  • Angiotensin II treatment: 0.5-2μM for 24-72h in endothelial cells

• Analysis Timeline:

  • Establish a timeline of SESN2 expression (24h, 48h, 72h post-stress)

  • Monitor correlating stress markers (e.g., HO-1, Bip/GRP78, CHOP)

  • Assess downstream effectors (Nrf2, Keap1, LC3-II, p62)

• Functional Assessments:

  • Cell viability assays (MTT, LDH release)

  • ROS measurement using DCFH-DA fluorescence

  • Analysis of SESN2-dependent signaling through Western blot for AMPK, mTOR pathways

  • ER morphology examination by transmission electron microscopy

Research has shown that SESN2 expression increases time-dependently following stress induction, with peak expression often occurring 48-72 hours after initial exposure , suggesting experimental timelines should extend beyond immediate early responses.

What are the critical considerations when examining SESN2's role in autophagy regulation?

SESN2's role in autophagy regulation involves complex signaling patterns:

• Experimental Markers:

  • Monitor LC3-II/LC3-I ratio as autophagy indicator

  • Track p62 degradation (decreases with autophagy activation)

  • Assess AMPK phosphorylation status (activated by SESN2)

  • Examine mTORC1 inhibition (downstream effect of SESN2)

• Modulatory Approaches:

  • Autophagy inhibition: chloroquine (10μM) or bafilomycin A1

  • SESN2 manipulation: siRNA knockdown and overexpression

  • Pathway intervention: compound C (AMPK inhibitor), rapamycin (mTOR inhibitor)

• Analytical Methods:

  • Fluorescence microscopy for LC3 puncta formation

  • Western blotting for autophagy markers

  • Co-immunoprecipitation to detect SESN2-AMPK interaction

  • Transmission electron microscopy for autophagic vesicle visualization

Research demonstrates that SESN2 knockdown inhibits autophagic flux with decreased LC3-II and increased p62 levels, while affecting downstream pathways involving Keap1/Nrf2 signaling . This suggests SESN2 antibodies are valuable tools for monitoring this regulatory axis.

How can SESN2 antibody be used to study the interplay between SESN2 and Nrf2 signaling?

The SESN2-Nrf2 relationship represents a critical research area:

• Experimental Design Elements:

  • Sequential analysis of SESN2 and Nrf2 expression timing (mRNA and protein)

  • Nuclear vs. cytoplasmic fractionation to track Nrf2 translocation

  • Keap1 protein level assessment (inversely correlates with Nrf2 activation)

  • SESN2 manipulation through genetic approaches

• Mechanistic Investigation:

  • Assess p62-dependent autophagy using p62 siRNA alongside SESN2 modulation

  • Monitor Keap1 degradation through immunofluorescence and Western blotting

  • Use proteasome inhibitors (MG132) to distinguish between degradation mechanisms

  • Apply chloroquine to block autophagy and examine effects on Nrf2 pathway

• Functional Readouts:

  • Measure Nrf2 target genes (HO-1, NQO1) expression

  • Assess antioxidant capacity through ROS measurements

  • Evaluate cell survival under oxidative challenge

  • Quantify nuclear Nrf2 levels using SESN2-FITC co-localization

Research has demonstrated that SESN2 upregulation further reduces Keap1 protein levels and enhances nuclear translocation of Nrf2, while SESN2 silencing increases Keap1 and decreases Nrf2 activity . These findings position SESN2 as an upstream regulator of the Nrf2 antioxidant response.

What are common technical issues with SESN2 antibody FITC conjugates and how can they be resolved?

Researchers may encounter several challenges when working with SESN2-FITC antibodies:

• High Background Signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution: Increase blocking time (1-2 hours), optimize antibody dilution (start with 1:200 and adjust), add 0.1% Tween-20 to washing buffer, and include additional washing steps

• Weak or No Signal:

  • Cause: Low SESN2 expression, over-fixation, inappropriate permeabilization, or antibody degradation

  • Solution: Use positive controls with known SESN2 expression, reduce fixation time, optimize permeabilization, and ensure proper antibody storage (-20°C with minimal freeze-thaw cycles)

• Non-specific Binding:

  • Cause: Cross-reactivity with other proteins or insufficient washing

  • Solution: Pre-adsorb antibody with cell/tissue lysates, increase wash duration and frequency, and validate with SESN2 knockout or knockdown samples

• Photobleaching:

  • Cause: Excessive exposure to light or inappropriate mounting medium

  • Solution: Minimize light exposure during procedures, use anti-fade mounting media, and image samples promptly after preparation

When conducting co-localization studies with SESN2-FITC and ER markers, researchers have reported optimal results with shorter fixation times (10 minutes) and careful antibody titration to minimize background interference .

How should SESN2 antibody experiments be designed when studying tissues or cells with differential SESN2 expression?

SESN2 expression varies across tissues and cellular stress states, requiring tailored experimental approaches:

• Baseline Expression Assessment:

  • Perform preliminary quantification of SESN2 across experimental samples

  • Adjust antibody concentration based on expression level (higher dilutions for high-expressing samples)

  • Include gradient standards or calibrators for accurate comparisons

• Tissue-Specific Considerations:

  • For liver tissue (high SESN2 expression): Use higher antibody dilutions (1:1000) and shorter incubation times

  • For immune cells (variable expression): Pre-test antibody on isolated cell populations

  • For endothelial cells: Consider AngII pre-treatment to upregulate SESN2 for easier detection

• Experimental Controls:

  • Include tissue/cell-matched SESN2 knockout controls when possible

  • Use reference tissues with known expression levels

  • Apply siRNA knockdown in a subset of samples to validate signal specificity

Studies examining SESN2 in dendritic cells have successfully employed lentiviral SESN2 overexpression and siRNA knockdown approaches to create controls with varying expression levels, allowing more precise antibody optimization .

How can I quantitatively analyze SESN2 expression changes in complex experimental models?

Quantitative analysis of SESN2 requires rigorous methodological approaches:

• Western Blot Quantification:

  • Normalize SESN2 signal to loading controls (β-actin, GAPDH)

  • Use gradient loading to ensure linear detection range

  • Apply densitometric analysis with appropriate software (ImageJ)

  • Present data as fold-change relative to control conditions

• Flow Cytometry Analysis:

  • Establish negative and positive controls for gating

  • Present data as Mean Fluorescence Intensity (MFI) or percentage of positive cells

  • Include Fluorescence Minus One (FMO) controls to set thresholds

  • Consider dual staining with apoptosis markers to correlate SESN2 with cell viability

• Immunofluorescence Quantification:

  • Capture images using standardized exposure settings

  • Analyze multiple fields (>5) per condition

  • Measure integrated density or mean fluorescence intensity

  • Apply background subtraction consistently across samples

Research examining SESN2 in stress responses has demonstrated the importance of time-course experiments, with measurements at multiple time points (24h, 48h, 72h) revealing distinct expression patterns that might be missed in single time-point analyses .

How can SESN2 antibody be used to investigate SESN2's role in immune cell function?

SESN2 plays crucial roles in immune cells, particularly dendritic cells (DCs) and B cells:

• Dendritic Cell Applications:

  • Track SESN2 expression during DC maturation and activation

  • Investigate SESN2's protective role against endoplasmic reticulum stress

  • Examine SESN2-ATF4 interactions through co-immunoprecipitation

  • Assess impact on pyroptosis markers (NLRP3, cleaved caspase-1, GSDMD-N)

• B Cell Research:

  • Monitor SESN2's involvement in IL-4-induced IgE class switching

  • Analyze SESN2 influence on germline ε transcript (GLTε) expression

  • Examine AMPK pathway activation in relation to SESN2 levels

  • Assess impact on surface IgE expression and IgE production

• Methodological Approaches:

  • Flow cytometry for surface marker and SESN2 co-expression

  • RT-PCR for transcriptional analysis of immune-related genes

  • ChIP assays to investigate transcription factor binding to SESN2 promoter

  • Functional assays (T cell activation, cytokine production, antigen presentation)

Research has demonstrated that SESN2 deficiency in mouse models results in decreased OVA-specific IgE production and reduced IgE class switching, suggesting SESN2 as a potential therapeutic target in allergic diseases .

What are the critical considerations when using SESN2 antibody to study metabolic regulation?

SESN2's role in metabolic regulation requires specialized experimental approaches:

• Hepatic Metabolism Studies:

  • Assess SESN2 expression in relation to insulin signaling components

  • Monitor AKT phosphorylation status (Thr308 and Ser473) following SESN2 manipulation

  • Examine SESN2's impact on FoxO1 phosphorylation and gluconeogenic gene expression

  • Investigate PI3K activity in SESN2-deficient versus wild-type liver samples

• Insulin Resistance Models:

  • Use hyperinsulinemic-euglycemic clamp studies for whole-body insulin sensitivity

  • Measure glucose infusion rate and hepatic glucose production

  • Assess SESN2 expression levels in different metabolic tissues

  • Examine IRS-1 tyrosine and serine phosphorylation patterns

• Energy Stress Responses:

  • Apply 2-deoxyglucose (2-DG) to induce energetic stress

  • Monitor SESN2 expression in relation to PI3K/Akt pathway activation

  • Use PI3K inhibitors (LY294002) to examine pathway dependence

  • Assess SESN2's protective role against energetic stress-induced apoptosis

Research has shown that SESN2-deficient mice exhibit reduced hepatic insulin sensitivity, with impaired insulin-stimulated AKT phosphorylation and increased gluconeogenic gene expression, positioning SESN2 as a critical regulator of hepatic metabolism .

How can researchers investigate SESN2's role in endothelial cell function using SESN2 antibody?

Endothelial cell studies with SESN2 require specialized approaches:

• Experimental Models:

  • Human umbilical vein endothelial cells (HUVECs) as primary model

  • Angiotensin II (0.5-2μM) treatment to induce SESN2 expression

  • Time-course experiments (24h, 48h, 72h) to capture expression dynamics

  • Endothelial progenitor cells (EPCs) for regeneration studies

• Functional Assessments:

  • Cell viability (MTT assay) and cytotoxicity (LDH release) measurements

  • ROS production quantification using DCFH-DA fluorescence

  • Oxidative stress marker (4-HNE) detection by immunofluorescence

  • Apoptotic markers (cleaved caspase-3, Bcl-2/Bax ratio) by Western blot

• Mechanistic Investigations:

  • SESN2 knockdown using siRNA approaches

  • Assessment of Nrf2/Keap1 pathway components

  • Analysis of autophagy markers (LC3-II, p62)

  • Examination of antioxidant response elements

Research has demonstrated that SESN2 knockdown exacerbates Angiotensin II-induced endothelial cell damage by increasing oxidative stress and apoptosis, while enhancing ROS production and LDH release, suggesting SESN2 as a potential therapeutic target for endothelial protection .

What approaches can be used to study SESN2 promoter regulation and transcriptional control?

Understanding SESN2 transcriptional regulation involves specialized techniques:

• Promoter Analysis Methods:

  • Luciferase reporter assays with wild-type and mutant SESN2 promoter constructs

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding

  • EMSA (Electrophoretic Mobility Shift Assay) to confirm direct DNA-protein interactions

  • Deletion analysis to identify critical promoter regions

• Transcription Factor Studies:

  • Focus on C/EBPβ binding to SESN2 promoter under stress conditions

  • Examine PERK-dependent transcription factor activation

  • Investigate ATF4, ATF6, and XBP1 roles in stress-induced SESN2 expression

  • Apply transcription factor knockdown approaches to validate regulatory relationships

• Stress-Responsive Elements:

  • Analyze endoplasmic reticulum stress-responsive elements in SESN2 promoter

  • Examine hypoxia-responsive elements for stress-induced expression

  • Investigate upstream sequences (approximately 2,000 bp spanning -2,396 to -214)

  • Use site-directed mutagenesis to confirm functional binding sites

Research has demonstrated that C/EBPβ binds to the SESN2 promoter under stress conditions, with ChIP assays confirming enrichment of SESN2 promoter in C/EBPβ-bound complexes. Additionally, luciferase assays with wild-type versus mutant SESN2 promoters have revealed functional significance of these binding events .

What are emerging applications of SESN2 antibody in therapeutic target validation?

SESN2 shows promise as a therapeutic target in several pathological conditions:

• Metabolic Disorders:

  • SESN2 activation as potential approach for hepatic insulin resistance

  • Development of small molecule SESN2 inducers for metabolic syndrome

  • Monitoring SESN2 levels as biomarker for metabolic disease progression

• Inflammatory and Immune Disorders:

  • SESN2 targeting for IgE-mediated allergic diseases

  • SESN2 enhancement for protection against sepsis-induced dendritic cell dysfunction

  • Development of immune cell-specific SESN2 modulators

• Cardiovascular Applications:

  • SESN2 upregulation for endothelial protection

  • Prevention of AngII-induced vascular damage

  • Monitoring SESN2 as biomarker for endothelial dysfunction

The research potential in these areas relies on antibody-based validation of target engagement and pathway modulation. SESN2 antibodies are essential tools for confirming that therapeutic interventions successfully alter SESN2 expression or function in relevant cell types and tissues .

How can researchers integrate SESN2 antibody approaches with omics technologies?

Integration of SESN2 research with advanced omics approaches offers new research dimensions:

• Proteomics Integration:

  • Immunoprecipitation with SESN2 antibody followed by mass spectrometry to identify interaction partners

  • Phosphoproteomics to map SESN2-dependent signaling networks

  • Targeted proteomics to quantify SESN2 pathway components across experimental conditions

  • Spatial proteomics to map SESN2 subcellular localization dynamics

• Transcriptomics Applications:

  • RNA-seq following SESN2 modulation to identify downstream gene networks

  • Single-cell transcriptomics to identify cell populations with differential SESN2 expression

  • Analysis of SESN2-dependent transcriptional programs under stress conditions

  • Integration with ChIP-seq data to map transcription factor networks regulating SESN2

• Multi-omics Approaches:

  • Correlate SESN2 protein levels with transcriptional and metabolic changes

  • Integrate phosphoproteomics with metabolomics to understand SESN2's impact on cellular energetics

  • Develop computational models of SESN2-regulated networks based on multi-omics data