ESYT2 Antibody, FITC conjugated

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

ESYT2, also known as FAM62B, is an extended synaptotagmin-like protein that plays a critical role in membrane contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM). These ER-PM contact sites are essential for various cellular processes including lipid transfer, calcium signaling, and cell adhesion. Research has implicated ESYT2 in extracellular signal transduction and cell adhesion functions . The protein is encoded on chromosome 12 and is part of a family of proteins that includes ESYT1 and ESYT3, which have tissue-specific expression patterns . ESYT2's membrane localization makes it particularly valuable for studying ER-PM interactions in different cell types and under various physiological conditions.

What are the technical specifications of ESYT2/FAM62B Polyclonal Antibody with FITC conjugation?

The ESYT2/FAM62B Polyclonal Antibody with FITC conjugation is typically derived from rabbit hosts using KLH-conjugated synthetic peptides from human ESYT2/FAM62B protein (immunogen range: 801-921/921) . The antibody has the following specifications:

How does FITC conjugation impact antibody functionality and application?

• FITC-conjugated antibodies are sensitive to light exposure, which gradually diminishes fluorescence intensity .

• The conjugation process may slightly affect the antibody's binding affinity compared to unconjugated versions.

• FITC has an excitation maximum at approximately 495 nm and emission maximum at approximately 520 nm, requiring appropriate filter sets for visualization.

• Direct conjugation eliminates potential cross-reactivity issues associated with secondary antibodies, particularly important in multi-labeling experiments.

What are the validated applications for ESYT2 Antibody, FITC conjugated?

The ESYT2/FAM62B polyclonal antibody with FITC conjugation has been validated for multiple experimental applications :

• Western Blotting (WB) - For detecting ESYT2 protein in cell and tissue lysates

• Immunofluorescence on paraffin-embedded tissues (IF/IHC-P) - For localizing ESYT2 in fixed tissue sections

• Immunofluorescence on frozen tissue sections (IF/IHC-F) - For detecting ESYT2 in cryopreserved samples

• Immunocytochemistry (ICC) - For visualizing ESYT2 localization in cultured cells

These applications leverage the antibody's specificity for ESYT2/FAM62B and the direct visualization capabilities provided by the FITC conjugation.

What is the recommended protocol for immunofluorescence using FITC-conjugated ESYT2 antibody?

For optimal results with immunofluorescence using FITC-conjugated ESYT2 antibody, follow this methodological approach based on standard protocols for FITC-conjugated antibodies :

• Sample Preparation:

  • Seed cells on appropriate coverslips or glass-bottomed plates

  • For tissue sections, prepare paraffin-embedded or frozen sections as needed

• Fixation and Permeabilization:

  • Fix cells using 4% paraformaldehyde in PBS for 10 minutes at room temperature

  • Wash thoroughly with PBS

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Wash again with PBS

• Blocking:

  • Incubate with blocking buffer (10% FBS, 1% BSA, 0.1% Triton X-100, and 0.01% NaN₃ in PBS) for 1 hour at room temperature

• Antibody Incubation:

  • Dilute the FITC-conjugated ESYT2 antibody (recommended starting dilution 1:500) in blocking buffer

  • Incubate overnight at 4°C in a humidity chamber protected from light

  • Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Counterstaining (optional):

  • Counterstain nuclei with DAPI if desired

  • For multi-labeling experiments, use antibodies with compatible fluorophores

• Mounting and Imaging:

  • Mount with appropriate anti-fade mounting medium

  • Image using fluorescence microscopy with appropriate FITC filter sets

  • For optimal results in membrane contact site studies, consider using total internal reflection fluorescence (TIRF) microscopy

How should researchers optimize FITC-conjugated ESYT2 antibody dilutions for different experimental applications?

Optimizing antibody dilution is crucial for balancing specific signal and background. For FITC-conjugated ESYT2 antibody:

• Initial Titration Series:

  • For immunofluorescence: Start with 1:500 dilution as recommended, then test 1:250, 1:500, 1:1000, and 1:2000

  • For Western blotting: Begin with 1:1000 dilution as referenced for non-conjugated ESYT2 antibody , then test 1:500 and 1:2000

• Sample-Specific Considerations:

  • Cell lines with high ESYT2 expression (e.g., lung, spleen, testis, stomach tissues) may require higher dilutions

  • Primary cells versus cell lines may require different optimal dilutions

  • Fixed versus live cell applications may have different requirements

• Application-Specific Optimization:

  • Western blotting may require different dilutions than immunofluorescence

  • TIRF microscopy focusing on membrane contacts may benefit from higher antibody concentrations than wide-field imaging

  • Always include positive and negative controls when optimizing dilutions

How do endoplasmic reticulum-plasma membrane (ER-PM) contact sites impact ESYT2 localization and experimental design?

ESYT2 is involved in establishing and maintaining ER-PM contact sites, which has significant implications for experimental design :

• Membrane Contact Site Gradients:

  • Recent research has demonstrated that ER-PM contacts form gradients within cells

  • These gradients appear to direct key cellular processes

  • When studying ESYT2, researchers should consider its distribution across these gradients rather than simple presence/absence in subcellular fractions

• Visualization Techniques:

  • Total internal reflection fluorescence (TIRF) microscopy is particularly valuable for visualizing ESYT2 at ER-PM contact sites

  • This technique captures signals from the evanescent field, which extends only 100-200 nm from the coverslip surface

  • Using TIRF with FITC-conjugated ESYT2 antibody enables high-contrast imaging of membrane contact sites

• Co-localization Studies:

  • Consider co-staining with ER markers (e.g., SEC61β) and PM markers (e.g., CAAX-tagged fluorophores)

  • For advanced studies, cryo-electron tomography (cryo-ET) can provide nanometer-resolution of contact sites

How does tissue-specific expression of ESYT2 influence experimental design and interpretation?

ESYT2 expression varies significantly across tissues, which has important implications for experimental design :

• Tissue Expression Profile:

  • ESYT2 mRNA is predominantly detected in lung, spleen, testis, and stomach

  • Lower expression levels are found in other tissues

  • This expression pattern overlaps significantly with ESYT1, except in testis

• Experimental Implications:

  • Cell line selection should account for natural ESYT2 expression levels

  • RPE-1, HUVEC/TERT2, and BJ-5ta cells have been successfully used for studying ESYT2

  • When studying ESYT2 in tissues or cell types with low endogenous expression, sensitivity limitations must be considered

• Knockout Studies Interpretation:

  • Research has shown that loss of ESYT2 (and ESYT3) does not affect mouse viability

  • Genetic compensation or functional redundancy with ESYT1 may mask phenotypes

  • When designing knockout or knockdown experiments, consider potential compensatory mechanisms

What controls should be included when using FITC-conjugated ESYT2 antibody in advanced research applications?

For rigorous scientific validation, include these controls when working with FITC-conjugated ESYT2 antibody:

• Positive Controls:

  • Tissues known to express high ESYT2 levels (lung, spleen, testis, stomach)

  • Cell lines with confirmed ESYT2 expression

  • Overexpression systems with tagged ESYT2 constructs

• Negative Controls:

  • ESYT2 knockout or knockdown samples

  • Isotype control antibodies (rabbit IgG-FITC) to assess non-specific binding

  • Secondary antibody-only controls (for comparison with non-conjugated primary antibodies)

• Specificity Controls:

  • Pre-absorption with immunizing peptide to confirm binding specificity

  • Western blot validation to confirm molecular weight of detected protein

  • Multiple antibodies targeting different epitopes of ESYT2 to confirm localization patterns

How can researchers address photobleaching issues with FITC-conjugated antibodies?

FITC is particularly susceptible to photobleaching, which can limit experimental duration and quantitative analysis . To address this:

• Preventative Measures:

  • Minimize light exposure during all sample preparation steps

  • Use anti-fade mounting media containing anti-photobleaching agents

  • Store slides and samples in the dark at 4°C

  • Consider working under reduced ambient lighting conditions

• Imaging Strategies:

  • Use neutral density filters to reduce excitation intensity

  • Minimize exposure times and interval durations for time-lapse imaging

  • Capture FITC channel images first in multi-channel acquisition sequences

  • Consider deconvolution strategies to enhance signal from lower exposure images

• Alternative Approaches:

  • For long-term imaging or quantitative applications, consider antibodies conjugated to more photostable fluorophores (Alexa Fluor 488)

  • For fixed samples requiring archiving, document images promptly after preparation

What are potential causes and solutions for high background when using FITC-conjugated ESYT2 antibody?

High background can compromise signal-to-noise ratio in immunofluorescence experiments. Common causes and solutions include:

• Inadequate Blocking:

  • Extend blocking time to 2 hours or overnight at 4°C

  • Increase blocking agent concentration (try 15-20% FBS or 2-5% BSA)

  • Add 0.1-0.3M glycine to blocking buffer to reduce aldehyde-induced autofluorescence

• Non-specific Binding:

  • Increase antibody dilution (try 1:1000 or 1:2000)

  • Add 0.1-0.5% non-ionic detergent (Tween-20) to washing buffers

  • Pre-absorb antibody with acetone powder from non-target tissues

• Autofluorescence:

  • Treat samples with 0.1-1% sodium borohydride after fixation to reduce autofluorescence

  • Include 10mM cupric sulfate in 50mM ammonium acetate (pH 5.0) treatment

  • Use spectral unmixing during image acquisition or processing

• Fixation Issues:

  • Optimize fixation time (over-fixation can increase background)

  • Try alternative fixatives (methanol instead of paraformaldehyde)

  • Ensure complete permeabilization for intracellular epitopes

How should researchers interpret ESYT2 subcellular localization patterns?

ESYT2 localization requires careful interpretation based on its known roles at ER-PM contact sites :

• Expected Localization Patterns:

  • Primarily at the cell membrane, particularly at ER-PM contact sites

  • Punctate distribution rather than continuous membrane staining

  • Potential co-localization with ER markers at membrane-proximal sites

  • May form gradients across the cell rather than uniform distribution

• Interpretation Approaches:

  • Use co-localization with established markers (SEC61β for ER, CAAX for PM)

  • Compare distribution under normal conditions versus after calcium elevation (which may affect ESYT2 recruitment)

  • Consider three-dimensional distribution using z-stack imaging

  • Quantify intensity distributions relative to cell landmarks or gradients

• Comparative Analysis:

  • Compare ESYT2 patterns with related proteins ESYT1 and ESYT3

  • Consider cell type-specific differences in localization patterns

  • Analyze changes in localization under different physiological or stress conditions

What quantitative methods are recommended for analyzing ESYT2 distribution at ER-PM contact sites?

For robust quantitative analysis of ESYT2 at ER-PM contact sites:

• Contact Site Quantification:

  • Measure density of ESYT2-positive puncta per unit membrane length

  • Calculate percentage of cell periphery containing ESYT2-positive contacts

  • Measure intensity profiles perpendicular to the plasma membrane to assess depth of contacts

• Gradient Analysis:

  • Develop line scan analyses across cellular axes to quantify ESYT2 distribution gradients

  • Use polar coordinate transformations to normalize cells of different shapes

  • Apply heat maps to visualize distributional differences across experimental conditions

• Co-localization Analysis:

  • Calculate Pearson's or Manders' correlation coefficients with ER and PM markers

  • Use object-based colocalization to identify bona fide contact sites

  • Apply super-resolution techniques (STORM, STED) for nanometer-scale analysis