SESN2 Antibody, Biotin conjugated

What is SESN2 and why is it an important research target?

SESN2 (Sestrin 2) is a stress-inducible metabolic regulator protein belonging to the sestrin family of PA26-related proteins. It plays crucial roles in:

• Functioning as an intracellular leucine sensor that negatively regulates the mTORC1 signaling pathway through the GATOR complex

• Protection against oxidative and genotoxic stresses

• Cell adaptation to limiting glucose conditions

• Regulating protein translation in response to endoplasmic reticulum stress

• Facilitating SQSTM1-mediated autophagic degradation of KEAP1

The protein has a calculated molecular weight of approximately 54 kDa, though the observed molecular weight in Western blot applications typically ranges from 54-60 kDa . SESN2's involvement in multiple cellular stress response pathways makes it a valuable target for research into metabolic disorders, cancer, and cellular stress responses .

What applications is a biotin-conjugated SESN2 antibody suitable for?

Biotin-conjugated SESN2 antibodies are versatile research tools applicable to multiple experimental techniques:

The biotin conjugation provides enhanced sensitivity through the strong biotin-streptavidin interaction, making these antibodies particularly valuable for detecting low-abundance SESN2 in complex samples .

What is the proper storage and handling protocol for biotin-conjugated SESN2 antibodies?

To maintain optimal antibody performance:

• Store at -20°C for long-term storage

• Aliquot upon first thaw to avoid repeated freeze-thaw cycles

• Some formulations contain glycerol (typically 50%) and can be stored at 2-8°C for 6 months after reconstitution

• Typical storage buffer includes PBS (pH 7.3-7.4), glycerol, and preservatives such as sodium azide (0.02%)

• Avoid exposure to light, particularly important for biotin-conjugated antibodies

Following these guidelines will help maintain antibody activity and specificity throughout your research project timeline .

What species reactivity can I expect with SESN2 antibodies?

SESN2 antibodies show variable cross-reactivity depending on the specific product:

When selecting an antibody for your research, verify the manufacturer's validated reactivity for your species of interest. The high conservation of SESN2 across mammalian species often allows for cross-reactivity, but this should be experimentally validated in your specific system .

How does SESN2 subcellular localization change under stress conditions, and how can this be effectively visualized?

SESN2 exhibits dynamic subcellular localization that changes in response to cellular stress conditions. Research has demonstrated:

• Under normal glucose conditions, SESN2 is predominantly located in the cell nucleus

• Upon glucose withdrawal, SESN2 shows substantial translocation to the cytoplasm

To effectively visualize these changes:

• Use immunofluorescence with the following protocol modifications:

  • Recommended dilution for IF/ICC: 1:50-1:500

  • For optimal visualization, perform glucose deprivation experiments (0-24 hours)

  • Compare with control conditions using confocal microscopy

  • Co-stain with nuclear markers (DAPI) and cytoplasmic markers

• For quantitative assessment:

  • Perform subcellular fractionation followed by Western blot

  • Compare nuclear vs. cytoplasmic fractions under different stress conditions

  • Use biotin-conjugated antibodies for enhanced sensitivity in detecting translocation events

This subcellular redistribution likely relates to SESN2's function in inhibiting glycolysis and promoting cell survival under nutrient-limited conditions .

What are the technical considerations for using biotin-conjugated SESN2 antibodies in multiplex immunoassays?

When incorporating biotin-conjugated SESN2 antibodies into multiplex assays:

• Endogenous biotin interference:

  • Endogenous biotin in samples can cause background issues

  • Pre-block samples with avidin/streptavidin to minimize interference

  • Include biotin-blocking steps in your protocol for tissue samples

• Multiplex compatibility:

  • When combining with other detection systems, ensure spectral separation

  • If using fluorescent streptavidin conjugates, select fluorophores with minimal overlap

  • Test for antibody cross-reactivity with other targets in your multiplex panel

• Signal amplification strategies:

  • Leverage the biotin-streptavidin system for signal enhancement

  • Consider using streptavidin-HRP or streptavidin-AP for enzymatic amplification

  • For fluorescence applications, use streptavidin conjugated to bright fluorophores

• Validation controls:

  • Include single-stained controls to assess bleed-through

  • Use isotype controls (rabbit IgG) to determine non-specific binding

  • Incorporate SESN2 knockdown/knockout samples as negative controls

These considerations will help maximize specificity and minimize artifacts when using biotin-conjugated SESN2 antibodies in complex multiplex experimental designs .

What methodological approaches can detect SESN2 interactions with mRNA and other proteins?

SESN2 has been shown to interact with various mRNAs and proteins. To investigate these interactions:

• For SESN2-mRNA interactions:

  • RNA immunoprecipitation (RIP) assays have successfully detected SESN2 binding to HK2 mRNA

  • Protocol recommendations:

    • Use biotin-conjugated SESN2 antibody for pulldown

    • Follow with RT-qPCR to identify bound transcripts

    • Include controls for non-specific RNA binding

  • Research has shown SESN2 competes with IGF2BP3 for binding to the 3'-UTR region of HK2 mRNA

• For SESN2-protein interactions:

  • Co-immunoprecipitation (co-IP) using biotin-conjugated SESN2 antibodies

    • Reported successful application of SESN2 antibodies in co-IP experiments

    • Use appropriate lysis buffers that preserve protein-protein interactions

  • Proximity ligation assays for in situ detection of protein-protein interactions

  • Pull-down assays with biotin-tagged SESN2 followed by mass spectrometry

• For investigating SESN2 in stress granules:

  • Combined immunofluorescence approaches

  • Co-staining for stress granule markers and SESN2

  • Live-cell imaging with fluorescently tagged SESN2 and stress granule markers

These methodological approaches have revealed that SESN2 competes with IGF2BP3 for HK2 mRNA binding, which affects mRNA stability and plays a role in regulating glycolysis under stress conditions .

How can researchers validate SESN2 antibody specificity in experimental systems?

Rigorous validation of SESN2 antibody specificity is critical for reliable research outcomes:

• Genetic knockout/knockdown validation:

  • Compare antibody signal in SESN2 wild-type versus knockout/knockdown models

  • Published studies have utilized SESN2 knockdown as negative controls

  • Western blot analysis should show absence or significant reduction of the 54-60 kDa band

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare signal with and without peptide competition

  • Signal should be significantly reduced or abolished when the antibody is pre-absorbed

• Cross-reactivity assessment:

  • Test in multiple cell lines with varying SESN2 expression levels

  • Validated cell lines for SESN2 detection include HEK-293, HepG2, HeLa, K-562, and RAW 264.7

  • Compare results across different detection methods (WB, IHC, IF)

• Molecular weight confirmation:

  • Verify that detected bands match the expected molecular weight (54-60 kDa)

  • Check for potential post-translational modifications that might alter migration

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of SESN2

  • Concordant results across different antibodies increase confidence in specificity

These validation approaches ensure that experimental observations truly reflect SESN2 biology rather than antibody artifacts .

What are optimal experimental designs for studying SESN2's role in stress response pathways?

To effectively investigate SESN2's functions in stress response:

• Glucose deprivation studies:

  • Research has shown SESN2 upregulation during glucose withdrawal

  • Experimental design:

    • Culture cells in complete media vs. glucose-free media

    • Monitor SESN2 expression, subcellular localization, and downstream effects

    • Assess impact on glycolysis by measuring hexokinase 2 (HK2) levels

• Oxidative stress experiments:

  • SESN2 plays a role in oxidative stress protection

  • Treatment paradigms:

    • Hydrogen peroxide (H₂O₂) exposure (varying concentrations and timepoints)

    • Hypoxia/reoxygenation models

    • Measurement of ROS levels and cell viability

• mTORC1 signaling analysis:

  • SESN2 functions as a leucine sensor regulating mTORC1

  • Key experimental approaches:

    • Leucine deprivation and repletion experiments

    • Monitor phosphorylation of mTORC1 substrates (S6K, 4E-BP1)

    • Assess SESN2 interaction with GATOR complex components

• Comparative tissue analysis:

  • SESN2 expression varies across tissues

  • Validated tissues for SESN2 detection include:

    • Human liver and brain tissue

    • Human breast, ovary, and colon cancer tissues

    • Mouse kidney tissue

These experimental designs, coupled with appropriate controls and SESN2 detection methods, can effectively elucidate SESN2's multifaceted roles in cellular stress responses .

What technical challenges exist in detecting SESN2 in different sample types, and how can they be overcome?

Researchers face several technical challenges when detecting SESN2 across various sample types:

• Tissue-specific expression levels:

  • SESN2 expression varies considerably across tissues

  • Solution: Adjust antibody concentration based on expected expression level

    • For high-expressing tissues (liver): 1:5000-1:12000 dilution for WB

    • For moderate/low-expressing tissues: 1:500-1:2000 dilution

• Background issues in immunohistochemistry:

  • Biotin-conjugated antibodies may generate high background in biotin-rich tissues

  • Solutions:

    • Use specialized blocking solutions containing avidin/streptavidin

    • Optimize antigen retrieval methods (validated methods include):

      • TE buffer pH 9.0 (preferred)

      • Alternative: citrate buffer pH 6.0

• Detection in stress granule components:

  • SESN2 can localize to stress granules under certain conditions

  • Approach:

    • Use specialized fixation methods that preserve stress granule integrity

    • Consider detergent concentrations that maintain liquid-liquid phase separation

• Post-translational modifications:

  • SESN2 undergoes modifications that may affect antibody binding

  • Strategy:

    • Use antibodies targeting different epitopes

    • Consider phospho-specific antibodies for certain applications

• Cross-reactivity with SESN1 and SESN3:

  • SESN family proteins share sequence homology

  • Validation approach:

    • Perform parallel detection with SESN1/SESN3-specific antibodies

    • Include SESN2-specific peptide competition controls

By addressing these technical challenges with appropriate methodological modifications, researchers can achieve reliable and specific detection of SESN2 across diverse experimental systems .