ESYT2 Antibody, Biotin conjugated

What is ESYT2 and why is it significant in cellular research?

ESYT2 (Extended Synaptotagmin 2) is a membrane protein with 921 amino acid residues and a molecular mass of 102.4 kDa in humans. It belongs to the extended synaptotagmin protein family and plays crucial roles in endocytosis and lipid metabolism. ESYT2 has dual subcellular localization in both the endoplasmic reticulum (ER) and cell membrane, making it particularly important for studying membrane dynamics and cellular compartmentalization. It shows high expression levels in the cerebellum, suggesting specialized functions in neuronal tissues. Up to five different isoforms have been reported for this protein, indicating potential tissue-specific or condition-dependent expression patterns .

What are the main applications where biotin-conjugated ESYT2 antibodies demonstrate advantages over unconjugated versions?

Biotin-conjugated ESYT2 antibodies offer distinct advantages in several research applications. They excel in signal amplification workflows where detection sensitivity is crucial, such as in low-expression tissues or when studying subtle expression changes. The biotin-streptavidin system enables flexible detection through various reporter molecules (fluorophores, enzymes, gold particles) without requiring species-specific secondary antibodies. These conjugated antibodies are particularly valuable for multiplex immunodetection when combined with differently labeled primary antibodies. For proximity-based assays and protein-protein interaction studies, the biotin tag provides a consistent anchoring point. Additionally, biotin-conjugated ESYT2 antibodies are essential for specialized techniques like CITE-seq, where antibody-oligonucleotide conjugation via streptavidin creates protein-specific barcodes for single-cell multiomics .

How should I optimize protocols for detecting ESYT2 across different cellular compartments?

Optimizing ESYT2 detection across cellular compartments requires careful consideration of fixation and permeabilization methods that preserve both membrane and ER structures. A sequential fixation approach is recommended:

For immunofluorescence, include markers for both compartments (e.g., calnexin for ER, Na⁺/K⁺-ATPase for plasma membrane) to validate compartment-specific detection. When performing quantitative analysis, normalize ESYT2 signals to compartment-specific markers to account for fixation-induced variations in epitope accessibility .

What controls are essential when validating biotin-conjugated ESYT2 antibodies for research applications?

A comprehensive validation strategy for biotin-conjugated ESYT2 antibodies must include multiple controls:

• Biological controls:

  • Positive control: Tissue/cells with confirmed high ESYT2 expression (cerebellum)

  • Negative control: ESYT2 knockout/knockdown samples

  • Expression gradient: Series of samples with varying ESYT2 expression levels

• Technical controls:

  • Primary antibody omission: To assess streptavidin reporter background

  • Biotin blocking: Pre-treatment with avidin/biotin blocking kit to control endogenous biotin

  • Peptide competition: Pre-incubation with immunizing peptide should abolish specific binding

  • Unconjugated comparison: Parallel staining with unconjugated version of the same antibody clone

  • Cross-reactivity assessment: Testing in multiple species if cross-reactivity is claimed

• Detection system controls:

  • Streptavidin-only control: To assess endogenous biotin levels

  • Isotype control: Biotin-conjugated irrelevant antibody of same isotype

Results should show a single band at 102.4 kDa in Western blot, consistent subcellular localization patterns, and signal elimination in knockout controls .

How can I minimize background when using biotin-conjugated ESYT2 antibodies in tissues with high endogenous biotin?

Minimizing background in tissues with high endogenous biotin (such as brain, liver, and kidney) requires a systematic approach:

• Implement a sequential blocking strategy:

  • Block with unconjugated avidin (10-15 minutes)

  • Follow with excess free biotin (10-15 minutes)

  • Apply standard protein blocking solution (5% BSA or serum)

• Optimize antibody dilution:

  • Use higher dilutions than recommended for unconjugated antibodies

  • Determine optimal concentration through titration experiments (typically 1:500-1:2000)

• Modify detection methodology:

  • Use fluorophore-conjugated streptavidin with minimal spectral overlap with tissue autofluorescence

  • For enzymatic detection, consider using HRP-conjugated streptavidin with specialized substrates that generate minimal diffusible products

• Enhance washing protocols:

  • Extend washing time between steps (minimum 4 x 10 minutes)

  • Include 0.05-0.1% Tween-20 in washing buffers

  • Consider using higher salt concentration (up to 0.5M NaCl) in final washes

• For particularly problematic tissues, pre-treat sections with streptavidin-biotin quenching reagents before the standard blocking procedure .

How can biotin-conjugated ESYT2 antibodies be implemented in single-cell protein profiling techniques?

Implementing biotin-conjugated ESYT2 antibodies in single-cell protein profiling techniques like CITE-seq requires careful preparation of antibody-oligonucleotide conjugates. The process leverages the streptavidin-biotin interaction to link barcoded oligonucleotides to the antibodies:

• Preparation workflow:

  • Obtain 5' biotinylated oligonucleotides with cell barcodes (25 nmoles scale is sufficient)

  • Conjugate antibodies with streptavidin using commercial labeling kits (~2 streptavidin molecules per antibody)

  • Merge streptavidin-antibodies with biotinylated-oligos in PBS/0.5M NaCl (800 pmoles of biotinylated oligo)

  • Incubate overnight at room temperature

  • Purify conjugates using 50 kDa cutoff columns with multiple wash steps

• Validation steps:

  • Run gel electrophoresis to confirm successful conjugation

  • Test binding specificity in control cell lines

  • Optimize concentration to avoid cell aggregation

• Protocol adaptations:

  • Implement specialized blocking to prevent non-specific binding

  • Adjust cell staining concentration (typically 1-5 μg/ml)

  • Include isotype controls with matched oligonucleotide barcodes

This approach enables simultaneous measurement of ESYT2 protein expression alongside transcriptomic profiling at single-cell resolution, providing insights into correlation between protein and mRNA levels .

What approaches can resolve contradictory results when studying ESYT2 isoforms with different antibodies?

Resolving contradictory results when studying ESYT2 isoforms requires a systematic approach combining molecular and analytical techniques:

• Epitope mapping analysis:

  • Determine precise epitope locations for each antibody

  • Map epitopes against known isoform sequence variations

  • Identify antibodies that target isoform-specific regions versus conserved domains

• Validation using molecular techniques:

  • Implement isoform-specific knockdown (siRNA targeting unique exons)

  • Express recombinant isoforms individually as positive controls

  • Perform RT-PCR to correlate protein detection with isoform-specific transcript levels

• Advanced detection strategies:

  • Use high-resolution SDS-PAGE (8% gels) to separate closely sized isoforms

  • Implement 2D electrophoresis for separation by both size and charge

  • Consider mass spectrometry to identify isoform-specific peptides

• Statistical approaches for reconciling data:

  • Implement Bland-Altman analysis to quantify agreement between antibodies

  • Use principal component analysis to identify patterns across multiple antibody results

  • Develop computational models to deconvolute signals based on known isoform characteristics

When possible, use antibodies targeting different epitopes simultaneously in multiplexed detection to increase confidence in isoform identification .

How can biotin-conjugated ESYT2 antibodies be utilized to study protein-lipid interactions at membrane contact sites?

Studying ESYT2-lipid interactions at membrane contact sites with biotin-conjugated antibodies involves specialized approaches that leverage the biotin tag while preserving the delicate membrane architecture:

• In situ proximity analysis:

  • Implement proximity ligation assays (PLA) using biotin-conjugated ESYT2 antibodies paired with antibodies against lipid-binding domains

  • Perform FRET analysis using streptavidin-conjugated fluorophores as donors and lipid probes as acceptors

  • Apply super-resolution microscopy techniques (STORM, STED) for nanoscale spatial mapping of interactions

• Biochemical fractionation approaches:

  • Isolate membrane contact sites using subcellular fractionation

  • Perform immunoprecipitation with biotin-conjugated antibodies coupled to streptavidin beads

  • Analyze co-precipitated lipids using lipidomics approaches (LC-MS/MS)

• Dynamic interaction studies:

  • Track real-time changes in ESYT2-lipid associations during calcium fluctuations

  • Monitor redistribution following pharmacological manipulation of lipid composition

  • Quantify changes in ESYT2 localization during ER stress responses

• Reconstitution systems:

  • Create in vitro membrane systems with defined lipid compositions

  • Add purified ESYT2 protein followed by biotin-conjugated antibodies

  • Measure binding affinities and dynamics using surface plasmon resonance or microscale thermophoresis

These approaches provide complementary data on how ESYT2 interacts with specific lipid species and how these interactions change under different cellular conditions .

What are the most common technical issues with biotin-conjugated ESYT2 antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with biotin-conjugated ESYT2 antibodies:

For particularly challenging samples, consider using tyramide signal amplification (TSA) systems compatible with biotin-conjugated primary antibodies to enhance detection sensitivity while maintaining specificity .

How should researchers address potential epitope masking when detecting ESYT2 in fixed tissues?

Addressing epitope masking for ESYT2 detection requires a methodical approach to antigen retrieval and fixation optimization:

• Antigen retrieval methods comparison:

  • Heat-induced epitope retrieval (HIER): Test multiple buffers (citrate pH 6.0, Tris-EDTA pH 9.0, and Tris-HCl pH 10.0) at 95-98°C for 10-20 minutes

  • Enzymatic retrieval: Try proteolytic enzymes (proteinase K, trypsin) at controlled concentrations and times

  • Combination approach: Sequential application of HIER followed by mild enzymatic treatment

• Fixation protocol optimization:

  • Test progressive fixation times (10, 20, 30 minutes) to determine minimum effective time

  • Compare cross-linking fixatives (paraformaldehyde) with precipitating fixatives (methanol, acetone)

  • For challenging epitopes, use light fixation (1% PFA) followed by post-fixation after antibody binding

• Advanced solutions for persistent masking:

  • Screen multiple antibody clones targeting different ESYT2 epitopes

  • Consider freeze substitution techniques for optimal ultrastructure preservation

  • Apply protein denaturation steps (6M urea or 0.5% SDS with subsequent quenching) before antibody application

The effectiveness of these approaches varies based on tissue type and ESYT2 isoform distribution. Always validate optimized protocols across multiple specimens before proceeding with experimental analyses .

What methodological adaptations are necessary when transitioning from cell lines to primary tissues for ESYT2 detection?

Transitioning from cell lines to primary tissues for ESYT2 detection requires specific methodological adaptations:

• Fixation and processing modifications:

  • Reduce fixation time for tissues (typically 50-75% of cell line protocols)

  • Implement graded fixation for larger tissue specimens to ensure even penetration

  • Consider perfusion fixation for animal tissues to preserve in vivo architecture

• Detection system adjustments:

  • Increase antibody concentration by 1.5-2x compared to cell line protocols

  • Extend primary antibody incubation (overnight at 4°C or 48 hours for thick sections)

  • Use amplification systems like biotin-tyramide amplification for low abundance detection

• Background reduction strategies:

  • Implement tissue-specific blocking (add 10% serum from the tissue species)

  • Pre-absorb antibodies with acetone powder from relevant tissues

  • Include additional blocking steps for endogenous enzymes and biotin

• Validation approaches:

  • Compare staining patterns against in situ hybridization for ESYT2 mRNA

  • Use multiple antibodies targeting different ESYT2 epitopes

  • Validate using tissues from knockout models or with siRNA knockdown in ex vivo tissue cultures

These adaptations account for the greater complexity, reduced antigen accessibility, and higher background commonly encountered in primary tissues compared to cell lines .

How should researchers quantify and normalize ESYT2 expression data from different detection methods?

Proper quantification and normalization of ESYT2 expression data requires method-specific approaches:

• Western blot quantification:

  • Use calibrated standards of recombinant ESYT2 protein to establish a standard curve

  • Normalize to multiple housekeeping proteins (GAPDH, β-actin, α-tubulin)

  • Calculate relative expression using integrated density values rather than peak intensity

  • Apply ratio normalization between ESYT2 isoforms when studying isoform distribution

• Immunofluorescence quantification:

  • Implement automated image analysis with consistent thresholding across samples

  • Normalize to cell number or tissue area rather than total protein

  • Use Z-score normalization when comparing across multiple imaging sessions

  • For compartment-specific analysis, normalize to compartment volume using 3D reconstruction

• Flow cytometry analysis:

  • Utilize quantitative flow cytometry with calibrated beads to convert to molecules of equivalent soluble fluorochrome (MESF)

  • Apply compensation matrices to correct for spectral overlap in multiplex assays

  • Use median fluorescence intensity rather than mean for non-normal distributions

  • Normalize to isotype controls matched for biotin conjugation level

• Cross-platform data integration:

  • Transform datasets to comparable scales using rank-based normalization

  • Apply quantile normalization when integrating data from different detection methods

  • Implement Bayesian normalization approaches for small sample sizes

These methods ensure that ESYT2 expression data remains comparable across experimental conditions and detection platforms .

How can researchers differentiate between altered ESYT2 expression and redistribution in experimental models?

Differentiating between changes in ESYT2 expression levels versus subcellular redistribution requires complementary analytical approaches:

• Multi-method expression analysis:

  • Compare total protein levels (Western blot, ELISA) with transcript levels (qPCR, RNA-seq)

  • Implement pulse-chase labeling to distinguish between altered synthesis versus degradation

  • Use protein half-life measurements to account for stability changes

• Compartment-specific quantification:

  • Perform subcellular fractionation followed by Western blot analysis of each fraction

  • Use ratiometric imaging with compartment-specific markers in fixed samples

  • Implement live-cell imaging with photoactivatable ESYT2 constructs to track protein movement

• Statistical approaches for distinguishing patterns:

  • Apply coefficient of variation analysis across subcellular regions

  • Use Manders' overlap coefficient to quantify co-localization with compartment markers

  • Implement spatial point pattern analysis to detect clustering versus dispersal

• Advanced visualization techniques:

  • Create heatmaps of subcellular distribution patterns across treatment conditions

  • Generate 3D surface plots of intensity distributions to visualize subtle shifts

  • Use machine learning algorithms to classify distribution patterns independent of total intensity

These approaches provide complementary data that can distinguish between true expression changes and redistribution phenomena that might otherwise be misinterpreted in single-method studies .

What bioinformatic approaches can correlate ESYT2 expression with lipid metabolism pathways?

Integrating ESYT2 expression data with lipid metabolism requires sophisticated bioinformatic approaches:

• Pathway enrichment analysis:

  • Map ESYT2 co-expressed genes against specialized lipid metabolism databases (LIPID MAPS, LipidBank)

  • Perform gene set enrichment analysis (GSEA) using custom lipid metabolism gene sets

  • Apply topology-based pathway analysis to identify ESYT2's position in lipid regulatory networks

• Multi-omics integration strategies:

  • Implement canonical correlation analysis between transcriptomics, proteomics, and lipidomics datasets

  • Use partial least squares discriminant analysis (PLS-DA) to identify lipid species most strongly associated with ESYT2 expression

  • Apply Bayesian network analysis to model causality between ESYT2 levels and lipid pathway activities

• Visualization tools for complex associations:

  • Create correlation heatmaps between ESYT2 expression and lipid metabolites

  • Develop network visualizations with ESYT2 as a central node connected to lipid metabolism enzymes

  • Generate chord diagrams to illustrate relationships between ESYT2 domains and specific lipid binding profiles

• Machine learning approaches:

  • Implement random forest algorithms to identify non-linear relationships between ESYT2 and lipid pathways

  • Use support vector machines to classify samples based on combined ESYT2 expression and lipid profiles

  • Apply deep learning to predict functional consequences of ESYT2 alterations on lipid homeostasis

These bioinformatic strategies provide holistic understanding of how ESYT2 functions within the broader context of cellular lipid metabolism .

How can biotin-conjugated ESYT2 antibodies contribute to understanding neurodegenerative disorders?

Biotin-conjugated ESYT2 antibodies offer valuable insights into neurodegenerative pathophysiology through several specialized applications:

• Neuropathological analysis:

  • Perform comparative immunohistochemistry on post-mortem brain tissues from patients with Alzheimer's, Parkinson's, or other neurodegenerative conditions

  • Implement multiplexed fluorescence to co-localize ESYT2 with disease-specific markers (amyloid-β, tau, α-synuclein)

  • Quantify alterations in ESYT2 distribution at ER-PM contact sites in affected versus unaffected brain regions

• Mechanistic investigations:

  • Apply proximity ligation assays to detect altered interactions between ESYT2 and calcium signaling proteins

  • Track ESYT2 redistribution in primary neuronal cultures exposed to disease-relevant stressors

  • Monitor ESYT2-associated lipid transport alterations in models of lipid metabolism dysfunction

• Translational research applications:

  • Correlate ESYT2 expression patterns with clinical parameters (cognitive scores, disease duration)

  • Develop ESYT2-targeting therapeutic strategies based on identified dysfunction

  • Utilize ESYT2 as a biomarker for ER stress responses in cerebrospinal fluid or exosomes

The high expression of ESYT2 in cerebellum makes it particularly relevant for cerebellar ataxias and other movement disorders, while its role in ER-PM contact sites connects to calcium dysregulation common across neurodegenerative conditions .

What role does ESYT2 play in cellular responses to ER stress, and how can this be studied?

ESYT2's involvement in ER stress responses can be systematically investigated using biotin-conjugated antibodies:

• Temporal dynamics analysis:

  • Track ESYT2 redistribution during pharmacologically induced ER stress (tunicamycin, thapsigargin)

  • Monitor expression changes across ER stress time course (early, middle, late phases)

  • Correlate ESYT2 changes with canonical ER stress markers (BiP/GRP78, CHOP, XBP1 splicing)

• Functional relationship studies:

  • Examine calcium dynamics at ER-PM junctions using dual labeling of ESYT2 and calcium indicators

  • Assess lipid transfer alterations during ER stress using fluorescent lipid probes

  • Investigate ESYT2 phosphorylation status changes during stress using phospho-specific antibodies

• Interventional approaches:

  • Determine effects of ESYT2 knockdown/overexpression on cellular resilience to ER stress

  • Test pharmacological modulators of ER-PM contacts for effects on stress responses

  • Evaluate ESYT2 mutations associated with altered calcium sensing on ER stress susceptibility

• Disease-relevant models:

  • Study ESYT2 in cell types with high secretory burden (pancreatic β-cells, plasma cells)

  • Investigate ESYT2 in models of protein misfolding diseases (Alzheimer's, Parkinson's)

  • Examine ESYT2 dynamics in metabolic stress conditions (hyperlipidemia, insulin resistance)

These approaches collectively reveal how ESYT2 contributes to cellular adaptation to ER stress and how its dysfunction might contribute to pathological processes .

How can researchers investigate the potential role of ESYT2 in cancer biology?

Investigating ESYT2's role in cancer biology requires tailored experimental approaches:

• Expression analysis across cancer types:

  • Screen ESYT2 expression in tissue microarrays spanning multiple cancer types

  • Correlate expression with clinical parameters (stage, grade, survival)

  • Examine isoform-specific expression patterns that may have prognostic value

• Functional studies in cancer cells:

  • Modulate ESYT2 expression (knockdown/overexpression) and assess effects on:

    • Proliferation and apoptosis resistance

    • Migration and invasion capacity

    • Lipid metabolism alterations common in cancer

• Mechanistic investigations:

  • Study ESYT2's role in calcium signaling pathways frequently dysregulated in cancer

  • Investigate interactions with oncogenic signaling networks

  • Examine ESYT2-dependent lipid transport in relation to altered membrane composition in cancer cells

• Therapeutic targeting potential:

  • Evaluate ESYT2 as a biomarker for specific cancer subtypes

  • Assess vulnerability of cancer cells to ESYT2 modulation

  • Develop conjugated antibody-drug conjugates targeting cancer-specific ESYT2 epitopes

When conducting these studies, it's critical to use proper controls for endogenous biotin in cancer tissues, as many cancers exhibit altered biotin metabolism that can confound results with biotin-conjugated antibodies .