ESYT2 Antibody

What is ESYT2 and why is it studied in research?

ESYT2 (Extended synaptotagmin 2) is a member of the extended synaptotagmin protein family involved in endocytosis and lipid metabolism. In humans, the canonical protein consists of 921 amino acid residues with a molecular weight of approximately 102.4 kDa . ESYT2 is primarily localized to the endoplasmic reticulum (ER) and cell membrane, where it plays a crucial role in the formation of ER-plasma membrane junctions and their regulation in response to calcium signaling . The protein is widely expressed throughout the body with particularly high expression levels in the cerebellum . Research interest in ESYT2 has grown significantly due to its important roles in membrane contact sites and cellular calcium homeostasis, making ESYT2 antibodies essential tools for studying its expression, localization, and function in various physiological and pathological contexts.

What are the key differences between polyclonal and monoclonal ESYT2 antibodies for research applications?

Polyclonal ESYT2 antibodies, such as those available from OriGene and Proteintech, recognize multiple epitopes on the ESYT2 protein, providing higher sensitivity but potentially lower specificity . They are typically produced in rabbits and are useful for applications where signal amplification is crucial, such as detecting low-abundance ESYT2 in tissue samples.

Monoclonal ESYT2 antibodies, like the mouse monoclonal clone 4C3, recognize a single epitope, offering higher specificity but sometimes lower sensitivity . These antibodies provide more consistent results between experiments and are often preferred for quantitative applications where specific epitope recognition is critical.

The choice between polyclonal and monoclonal ESYT2 antibodies should be based on:

• Experimental application (WB, IHC, IF, etc.)

• Required specificity vs. sensitivity

• Target isoform detection needs (polyclonals may detect multiple isoforms)

• Cross-reactivity concerns with related proteins

For experiments requiring precise quantification or where background is problematic, monoclonal antibodies like those from Proteintech (68374-1-Ig) often provide cleaner results . For applications requiring detection of ESYT2 across multiple species or maximizing signal, polyclonal antibodies may be advantageous .

What are the common ESYT2 protein isoforms, and how do antibodies differ in detecting them?

ESYT2 exists in up to five different isoforms resulting from alternative splicing events, which can complicate antibody selection for specific research applications . The canonical isoform has 921 amino acids, but researchers should be aware that different ESYT2 antibodies may have varying affinities for different isoforms.

Most commercially available ESYT2 antibodies are designed against regions common to multiple isoforms. For example, Proteintech's polyclonal antibody (24385-1-AP) can detect ESYT2 at 90-100 kDa in Western blot applications across various human cell lines, including K-562, HeLa, and LNCaP cells . When selecting an ESYT2 antibody, researchers should carefully review the immunogen information to determine which isoforms an antibody is likely to detect.

If isoform-specific detection is required, researchers may need to:

• Select antibodies raised against isoform-specific epitopes

• Use complementary techniques like RT-PCR to verify isoform expression

• Consider knockout/knockdown validation experiments to confirm specificity

The observed molecular weight variations (between 90-100 kDa) in Western blot results may reflect different isoforms or post-translational modifications of ESYT2 .

How should I optimize Western blot protocols for detecting ESYT2 protein?

Optimizing Western blot protocols for ESYT2 detection requires careful consideration of several factors based on the protein's characteristics and the specific antibody being used:

• Sample preparation: For ESYT2, which localizes to both ER and plasma membranes, use lysis buffers containing mild detergents like RIPA buffer to effectively solubilize membrane-associated proteins .

• Gel percentage selection: Since ESYT2 has a molecular weight of 90-100 kDa, use 8-10% SDS-PAGE gels for optimal resolution in this range .

• Transfer conditions: For large proteins like ESYT2, longer transfer times or semi-dry transfer systems may improve transfer efficiency.

• Blocking and antibody dilutions:

  • For Proteintech's polyclonal antibody (24385-1-AP): Use 1:2000-1:16000 dilution

  • For Proteintech's monoclonal antibody (68374-1-Ig): Use 1:5000-1:50000 dilution

• Positive controls: Include lysates from cells known to express ESYT2, such as HeLa, K-562, LNCaP, A549, U2OS, HEK-293, Jurkat, or HL-60 cells .

Example optimization protocol:

• Start with 25-50 μg of total protein per lane

• Run on 8% SDS-PAGE gel

• Transfer to PVDF membrane (better for high MW proteins)

• Block with 5% non-fat milk in TBST for 1 hour

• Incubate with primary antibody (start at manufacturer's recommended dilution)

• Visualize using appropriate secondary antibody and detection system

Always include positive and negative controls, and consider running gradient dilutions of antibodies to determine optimal signal-to-noise ratio for your specific samples.

What are the recommended protocols for immunohistochemistry (IHC) using ESYT2 antibodies?

For optimal immunohistochemistry results with ESYT2 antibodies, follow these protocol recommendations:

• Tissue preparation and fixation:

  • Use 10% neutral buffered formalin for fixation (12-24 hours)

  • Embed in paraffin and section at 4-6 μm thickness

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0, heat-induced epitope retrieval

  • Alternative: Citrate buffer pH 6.0 if the above doesn't yield optimal results

• Antibody dilutions and incubation:

  • For Proteintech's polyclonal antibody (24385-1-AP): Use 1:250-1:1000 dilution

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Use a polymer-based detection system compatible with your primary antibody host species

  • Develop with DAB and counterstain with hematoxylin for standard brightfield visualization

• Positive control tissues:

  • Human intrahepatic cholangiocarcinoma tissue has shown positive results

  • Include cerebellum tissue samples when possible, as ESYT2 shows high expression in this tissue

A critical optimization step is antibody titration. Begin with the manufacturer's recommended dilution range and test serial dilutions to identify the optimal concentration that provides specific staining with minimal background. Always include positive and negative controls (omitting primary antibody) in each experiment to validate results.

How can I effectively use ESYT2 antibodies for immunofluorescence and confocal microscopy?

For high-quality immunofluorescence and confocal microscopy using ESYT2 antibodies, follow these detailed methodological guidelines:

• Cell preparation:

  • Grow cells on glass coverslips or chamber slides

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

  • For membrane proteins like ESYT2, mild permeabilization with 0.1-0.2% Triton X-100 (5-10 minutes) is recommended

• Blocking and antibody incubation:

  • Block with 5% normal serum from the same species as the secondary antibody

  • Incubate with primary ESYT2 antibody at optimized dilution (typically 1:100-1:500 for IF applications)

  • For co-localization studies, select compatible primary antibodies raised in different host species

• Visualizing ER-PM junctions:

  • Since ESYT2 localizes to ER-plasma membrane contact sites, co-staining with ER markers (e.g., calnexin, PDI) can help visualize these junctions

  • For high-resolution studies of membrane contacts, consider super-resolution techniques like STORM or STED microscopy

• Confocal settings optimization:

  • Use appropriate laser lines and filter sets for your fluorophores

  • Optimize pinhole settings (typically 1 Airy unit)

  • Capture Z-stacks when studying membrane contact sites to ensure accurate localization

• Controls and validation:

  • Include cells with ESYT2 knockdown or knockout as negative controls

  • Perform separate staining with just secondary antibodies to assess non-specific binding

  • Consider live-cell imaging with fluorescently tagged ESYT2 constructs to complement antibody-based detection

For dual or triple labeling experiments, carefully select fluorophores with minimal spectral overlap and acquire images sequentially rather than simultaneously to minimize bleed-through. This approach is particularly important when studying ESYT2's co-localization with other proteins at membrane contact sites.

Why might I observe multiple bands in Western blot when using ESYT2 antibodies?

Multiple bands in Western blot when using ESYT2 antibodies can result from several biological and technical factors that require careful interpretation:

• ESYT2 isoforms: Up to five different isoforms of ESYT2 have been reported, which may appear as distinct bands . The canonical isoform is 102.4 kDa, but other isoforms may have different molecular weights.

• Post-translational modifications: ESYT2 may undergo various post-translational modifications including phosphorylation, which can alter migration patterns. This may explain why the observed molecular weight (90-100 kDa) sometimes differs from the calculated weight .

• Protein degradation: ESYT2, as a large protein, may be susceptible to proteolytic degradation during sample preparation. To minimize this:

  • Add fresh protease inhibitors to lysis buffers

  • Keep samples cold during preparation

  • Avoid repeated freeze-thaw cycles

• Cross-reactivity: Some antibodies may cross-react with other extended synaptotagmin family members (ESYT1, ESYT3) that share structural similarities. Validate specificity using:

  • Knockout/knockdown controls

  • Peptide competition assays

  • Comparison with alternative antibody clones

• Non-specific binding: Particularly with polyclonal antibodies, non-specific binding can occur. To reduce this:

  • Optimize blocking conditions

  • Increase antibody dilution

  • Use freshly prepared buffers

Expected molecular weight patterns:

• Main ESYT2 band: 90-100 kDa

• Potential isoform bands: May appear above or below main band

• Degradation products: Multiple bands below expected size

For proper interpretation, compare your results with published literature and manufacturer's validation data. Consider using both monoclonal and polyclonal antibodies in parallel to confirm band identity.

How can I verify the specificity of my ESYT2 antibody?

Verifying ESYT2 antibody specificity is critical for generating reliable research data. Here are comprehensive methodological approaches to validate your antibody:

• Genetic knockdown/knockout validation:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of ESYT2

  • Compare antibody signal between wildtype and KD/KO samples

  • A specific antibody will show significantly reduced or absent signal in KD/KO samples

• Overexpression validation:

  • Transfect cells with tagged ESYT2 expression constructs

  • Perform parallel detection with anti-tag antibody and your ESYT2 antibody

  • Signals should overlap, confirming specificity

• Peptide competition assay:

  • Pre-incubate your antibody with the immunizing peptide/protein

  • Apply to duplicate samples alongside untreated antibody

  • Specific signals should be blocked in the peptide-treated sample

• Cross-validation with multiple antibodies:

  • Test multiple antibodies recognizing different ESYT2 epitopes

  • Compare staining patterns across techniques (WB, IF, IHC)

  • Consistent patterns across antibodies suggest specificity

• Mass spectrometry validation:

  • Perform immunoprecipitation with your ESYT2 antibody

  • Analyze precipitated proteins by mass spectrometry

  • ESYT2 should be among the top identified proteins

• Tissue/cell type expression pattern:

  • Compare antibody staining with known ESYT2 expression patterns

  • For example, cerebellum should show high expression levels

  • Expression patterns should match transcript data from resources like GTEx or Human Protein Atlas

Remember that ideal validation combines multiple approaches. Document your validation methods thoroughly when publishing results, as this enhances reproducibility and confidence in your findings.

What controls should I include when using ESYT2 antibodies in my experiments?

Including appropriate controls when working with ESYT2 antibodies is essential for experimental rigor and data interpretation. Here's a comprehensive guide to controls across different applications:

Western Blot Controls:

• Positive control: Include lysates from cells known to express ESYT2, such as:

  • HeLa cells

  • K-562 cells

  • LNCaP cells

  • A549 cells

  • HEK-293 cells

• Negative control:

  • ESYT2 knockdown/knockout cells

  • Cell lines with known low/no ESYT2 expression

  • Secondary antibody only to assess non-specific binding

• Loading control:

  • Housekeeping proteins (β-actin, GAPDH, tubulin)

  • Total protein stain (Ponceau S, REVERT)

Immunohistochemistry Controls:

• Positive control tissue:

  • Human intrahepatic cholangiocarcinoma tissue

  • Cerebellum (high ESYT2 expression)

• Negative controls:

  • Omit primary antibody

  • Isotype control (non-specific IgG from same species)

  • Peptide competition (pre-absorb antibody with immunizing peptide)

Immunofluorescence Controls:

• Specificity controls:

  • ESYT2 knockdown cells

  • Secondary antibody only

  • Peptide competition

• Co-localization controls:

  • Known markers for ER (calnexin, calreticulin)

  • Plasma membrane markers (Na+/K+ ATPase, WGA)

General Experimental Controls:

• Antibody titration: Test serial dilutions to determine optimal concentration

• Multiple antibody validation: Use antibodies from different sources/clones

• Multiple detection methods: Confirm findings across different techniques

Including these controls systematically will strengthen your experimental design and provide necessary context for interpreting ESYT2 antibody results, particularly when troubleshooting unexpected findings or presenting novel observations about ESYT2 localization or function.

How can ESYT2 antibodies be used to study ER-plasma membrane contact sites?

ESYT2 antibodies provide powerful tools for investigating ER-plasma membrane (ER-PM) contact sites through several advanced methodological approaches:

• Super-resolution microscopy techniques:

  • STORM, PALM, or STED microscopy can resolve ER-PM junctions (typically 10-30 nm wide)

  • Use ESYT2 antibodies in combination with ER markers (e.g., Sec61β) and PM markers

  • This approach can reveal the nanoscale organization of ESYT2 at membrane contact sites

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions between ESYT2 and potential binding partners

  • Requires antibodies raised in different species

  • Generates fluorescent signal only when proteins are within 40 nm

  • Useful for quantifying changes in ESYT2 interactions under different conditions (e.g., Ca2+ levels)

• Live-cell imaging combined with immunocytochemistry:

  • Transfect cells with fluorescently tagged ER/PM markers

  • Fix and perform immunostaining with ESYT2 antibodies

  • Track dynamic changes in ER-PM contacts followed by ESYT2 localization

• Electron microscopy techniques:

  • Immunogold labeling with ESYT2 antibodies for transmission electron microscopy

  • Correlative light and electron microscopy (CLEM) to precisely locate ESYT2 at ER-PM junctions

• Calcium imaging protocols:

  • Since ESYT2 is involved in Ca2+-dependent regulation of ER-PM junctions, combine ESYT2 immunostaining with calcium indicators

  • Monitor how calcium fluctuations affect ESYT2 distribution and ER-PM contacts

For quantitative analysis of ESYT2 at ER-PM junctions, establish clear criteria for defining contact sites and use automated image analysis tools to measure parameters such as:

• Number of ESYT2-positive contact sites per cell

• Contact site size distribution

• Colocalization coefficients with ER/PM markers

• Distance measurements between membrane components

This approach provides insights into how ESYT2 dynamically regulates the formation and maintenance of ER-PM junctions in response to cellular signaling events .

What are the best approaches for studying ESYT2 interactions with other proteins?

Investigating ESYT2 protein-protein interactions requires sophisticated methodological approaches. Here are the most effective techniques for studying ESYT2 interactome:

• Co-immunoprecipitation (Co-IP) with ESYT2 antibodies:

  • Use mild lysis conditions to preserve membrane protein interactions

  • For membrane proteins like ESYT2, consider crosslinking before lysis

  • Validate with reverse Co-IP using antibodies against suspected interaction partners

  • Western blot analysis of immunoprecipitates can confirm specific interactions

• Proximity-dependent labeling approaches:

  • BioID or TurboID: Fuse biotin ligase to ESYT2 to biotinylate proximal proteins

  • APEX2: Fuse peroxidase to ESYT2 for proximity-based labeling

  • These methods are particularly valuable for mapping interactions at membrane contact sites

  • Follow with mass spectrometry for unbiased interactome analysis

• FRET/FLIM analysis:

  • Förster Resonance Energy Transfer can detect direct protein interactions

  • Requires fluorescently tagged ESYT2 and potential partners

  • Confirms interactions with nanometer resolution in living cells

  • Particularly useful for studying calcium-dependent interactions

• Yeast two-hybrid membrane system:

  • Modified Y2H systems designed for membrane proteins

  • Can screen libraries to identify novel ESYT2 interaction partners

  • Validate hits with orthogonal methods in mammalian cells

• Pull-down assays with recombinant ESYT2 domains:

  • Express individual domains of ESYT2 (C2 domains, SMP domain)

  • Identify domain-specific interaction partners

  • Useful for mapping binding interfaces

• Crosslinking mass spectrometry (XL-MS):

  • Chemical crosslinking captures transient interactions

  • MS analysis identifies crosslinked peptides

  • Provides structural information about interaction interfaces

When designing interaction studies, consider the membrane-associated nature of ESYT2 and its calcium-responsive properties, which may influence binding partner selection under different cellular conditions .

How can ESYT2 antibodies be used in calcium signaling research?

ESYT2 antibodies can be powerful tools in calcium signaling research, particularly because ESYT2 functions in Ca2+-dependent regulation of ER-PM junctions. Here are methodological approaches for using these antibodies in calcium signaling studies:

• Temporal analysis of ESYT2 localization during calcium fluctuations:

  • Stimulate cells with calcium ionophores (ionomycin) or physiological stimuli

  • Fix cells at different time points after stimulation

  • Immunostain with ESYT2 antibodies to track redistribution

  • Quantify changes in ESYT2 localization at ER-PM junctions

• Combined calcium imaging and immunocytochemistry:

  • Load cells with calcium indicators (Fluo-4, Fura-2)

  • Record calcium responses to stimuli

  • Fix cells at specific phases of calcium response

  • Perform ESYT2 immunostaining to correlate calcium levels with ESYT2 dynamics

• ESYT2 translocation assays in different calcium conditions:

  • Prepare subcellular fractions (cytosolic, membrane, ER-enriched)

  • Western blot with ESYT2 antibodies to quantify distribution

  • Compare fractions from resting vs. calcium-stimulated cells

  • Correlate with known calcium-dependent proteins as positive controls

• Calcium-dependent protein interaction studies:

  • Perform co-immunoprecipitation with ESYT2 antibodies in:

    • Low calcium (+ EGTA) conditions

    • High calcium conditions

  • Identify differential binding partners by mass spectrometry

  • Validate calcium-sensitive interactions by Western blot

• ESYT2 phosphorylation state analysis:

  • Calcium often regulates proteins via phosphorylation changes

  • Use phospho-specific antibodies if available

  • Alternatively, perform immunoprecipitation with ESYT2 antibodies followed by phospho-staining

  • Compare phosphorylation states under different calcium conditions

• Functional studies combining calcium measurements with ESYT2 manipulation:

  • Measure store-operated calcium entry (SOCE) in:

    • ESYT2 knockdown cells

    • ESYT2 overexpressing cells

  • Use ESYT2 antibodies to confirm expression levels

  • Correlate ESYT2 levels with calcium signaling parameters

What are the most effective strategies for using ESYT2 antibodies in neuroscience research?

Given ESYT2's high expression in the cerebellum and its role in membrane contact sites that are crucial for neuronal function, ESYT2 antibodies offer valuable tools for neuroscience research . Here are methodological strategies optimized for neuronal systems:

• Immunohistochemical mapping of ESYT2 in neural tissues:

  • Use antigen retrieval with TE buffer pH 9.0 for optimal results in brain tissue

  • Compare ESYT2 distribution across brain regions, with special attention to cerebellum

  • Quantify expression levels in different neuron populations and glial cells

  • Combine with neuronal/glial markers for cell type-specific localization

• Synaptic localization studies:

  • Perform double immunofluorescence with ESYT2 antibodies and synaptic markers:

    • Pre-synaptic: synaptophysin, VAMP2

    • Post-synaptic: PSD-95, NMDA receptors

  • Use super-resolution microscopy to precisely map ESYT2 at synapses

  • Quantify co-localization coefficients in different synapse types

• Primary neuron culture applications:

  • Immunostain developing neurons at different stages (DIV1-21)

  • Track ESYT2 localization during neuronal maturation

  • Analyze distribution in dendrites, axons, and dendritic spines

  • Optimize fixation for neurons: 4% PFA with 4% sucrose works well

• Activity-dependent regulation studies:

  • Stimulate neurons (KCl, glutamate, electrical stimulation)

  • Fix at various time points post-stimulation

  • Immunostain for ESYT2 to detect activity-dependent redistribution

  • Combine with calcium imaging to correlate with calcium transients

• Neurodevelopmental analysis:

  • Examine ESYT2 expression across developmental stages

  • Compare expression patterns in developing vs. mature neurons

  • Correlate with synaptogenesis markers

• Techniques for enhanced neuronal imaging:

  • For cultured neurons: Optimize antibody penetration with longer incubation times

  • For brain slices: Use thinner sections (20-30 μm) with extended antibody incubation

  • For whole-mount preparations: Consider tissue clearing techniques compatible with immunolabeling

For quantitative analysis of ESYT2 in neurons, establish standardized imaging parameters and use automated analysis tools to measure:

• ESYT2 puncta density along neurites

• Distance from synaptic markers

• Changes in distribution following stimulation

• Co-localization with endoplasmic reticulum markers in dendritic spines

These approaches can provide insights into ESYT2's potentially specialized roles in neuronal calcium homeostasis, synaptic function, and neurodevelopment .

How can ESYT2 antibodies be used in studying lipid transfer at membrane contact sites?

ESYT2 is implicated in lipid transfer at ER-PM contact sites, making ESYT2 antibodies valuable tools for investigating these processes. Here are methodological approaches for studying ESYT2-mediated lipid dynamics:

• Combined lipid probes and ESYT2 immunostaining:

  • Use fluorescent lipid probes (NBD-lipids, BODIPY-lipids) to track lipid movement

  • Fix cells at different time points after lipid addition

  • Perform ESYT2 immunostaining to correlate lipid transfer with ESYT2 localization

  • Quantify co-localization between transferred lipids and ESYT2

• Lipidomics analysis with ESYT2 manipulation:

  • Immunoprecipitate ESYT2 using validated antibodies

  • Extract and analyze co-precipitated lipids by mass spectrometry

  • Compare lipid profiles between wildtype and ESYT2 knockdown/knockout samples

  • Identify lipid species specifically associated with ESYT2

• ESYT2 localization during phospholipid scrambling:

  • Induce phosphatidylserine exposure using calcium ionophores

  • Track PS exposure with annexin V labeling

  • Perform ESYT2 immunostaining

  • Analyze temporal relationship between PS exposure and ESYT2 redistribution

• Super-resolution microscopy of lipid domains and ESYT2:

  • Label specific lipid microdomains (e.g., cholesterol-rich regions with filipin)

  • Immunostain for ESYT2

  • Use STORM/STED microscopy to resolve nanoscale associations

  • Determine whether ESYT2 preferentially localizes to specific lipid environments

• FRAP (Fluorescence Recovery After Photobleaching) combined with immunocytochemistry:

  • Perform FRAP experiments with fluorescent lipid probes

  • Measure lipid mobility parameters

  • Fix cells and immunostain for ESYT2

  • Correlate lipid mobility with ESYT2 presence/absence

For quantitative analysis, develop metrics that measure:

• ESYT2 enrichment at sites of active lipid transfer

• Changes in lipid composition at ESYT2-positive membrane contact sites

• Kinetics of lipid movement in relation to ESYT2 expression levels

These approaches can reveal how ESYT2 contributes to lipid homeostasis through its roles at membrane contact sites, potentially providing insights into both physiological functions and disease mechanisms related to lipid dysregulation .

What methods can be used to study ESYT2 in disease models, and how do antibodies facilitate this research?

ESYT2 antibodies enable detailed investigation of this protein's role in various disease contexts. Here are methodological approaches for disease-focused ESYT2 research:

• Expression profiling in disease tissues:

  • Perform immunohistochemistry with ESYT2 antibodies on:

    • Tumor tissues (intrahepatic cholangiocarcinoma shows positive staining)

    • Neurodegenerative disease samples (given ESYT2's high expression in cerebellum)

    • Metabolic disease tissues (considering ESYT2's role in lipid metabolism)

  • Compare expression patterns with matched healthy controls

  • Quantify expression differences using digital pathology tools

• Cell stress response studies:

  • Induce ER stress with tunicamycin or thapsigargin

  • Track ESYT2 expression and localization using antibodies

  • Correlate with ER stress markers (BiP/GRP78, XBP1 splicing)

  • Analyze how ESYT2 distribution changes during stress resolution

• Calcium dysregulation models:

  • Use disease models characterized by calcium homeostasis disruption

  • Employ ESYT2 antibodies to assess:

    • Expression level changes

    • Subcellular redistribution

    • Interactions with calcium handling proteins

• Patient-derived cell models:

  • Obtain cells from patients with relevant disorders

  • Immunostain for ESYT2 to assess expression and localization

  • Compare with healthy donor cells

  • Correlate abnormalities with disease phenotypes

• Therapeutic target validation:

  • Test compounds that modulate ER-PM contacts

  • Use ESYT2 antibodies to monitor drug effects on:

    • Expression levels (by Western blot)

    • Subcellular distribution (by immunofluorescence)

    • Protein interactions (by co-immunoprecipitation)

• Disease-relevant functional assays:

  • Monitor calcium signaling in disease models with altered ESYT2 expression

  • Track lipid transfer defects using fluorescent lipid probes alongside ESYT2 immunostaining

  • Assess ER-PM contact site dynamics using live imaging followed by ESYT2 immunocytochemistry

When designing disease-focused experiments, consider tissue-specific expression patterns of ESYT2 and select appropriate antibody applications based on the specific research questions and disease mechanisms under investigation .

How should researchers validate and benchmark different ESYT2 antibodies for optimal experimental outcomes?

Systematic validation and benchmarking of ESYT2 antibodies is essential for generating reliable research data. Here's a comprehensive methodological framework:

• Multi-antibody comparative analysis:

  • Test multiple ESYT2 antibodies from different sources:

    • Polyclonal rabbit antibodies (e.g., Proteintech 24385-1-AP)

    • Monoclonal mouse antibodies (e.g., Proteintech 68374-1-Ig, Abnova clone 4C3)

  • Compare performance across applications (WB, IHC, IF)

  • Document sensitivity and specificity differences

• Application-specific validation matrix:

  • For Western blot: Compare detection limits and band patterns

  • For IHC: Compare staining patterns and background

  • For IF: Compare subcellular localization patterns

  • Create a structured scoring system for each parameter

• Genetic validation strategies:

  • ESYT2 siRNA knockdown: Test signal reduction for each antibody

  • ESYT2 CRISPR knockout: Confirm complete signal loss

  • ESYT2 overexpression: Verify signal increase

  • Document performance of each antibody in these validation tests

• Epitope mapping considerations:

  • Identify the epitope region for each antibody when available

  • Consider how epitope location might affect detection of:

    • Different ESYT2 isoforms

    • Post-translationally modified ESYT2

    • ESYT2 in protein complexes

  • Select antibodies targeting different epitopes for confirmation

• Reproducibility assessment:

  • Test lot-to-lot variation for each antibody

  • Evaluate performance across different sample preparations

  • Document consistency across multiple experiments

Example scoring matrix for ESYT2 antibody benchmarking:

This systematic benchmarking approach provides objective criteria for selecting the optimal ESYT2 antibody for specific experimental questions, ensuring more reliable and reproducible results across studies .

What are the advanced techniques for quantifying ESYT2 expression levels using antibody-based methods?

Accurate quantification of ESYT2 expression requires sophisticated antibody-based approaches beyond simple detection. Here are advanced methodological techniques:

• Quantitative Western blotting:

  • Use fluorescently-labeled secondary antibodies for wider linear dynamic range

  • Include recombinant ESYT2 protein standards at known concentrations

  • Normalize to total protein stains rather than single housekeeping proteins

  • Employ digital image acquisition and analysis software

  • Recommended dilutions for Proteintech antibodies:

    • Polyclonal 24385-1-AP: 1:2000-1:16000

    • Monoclonal 68374-1-Ig: 1:5000-1:50000

• Multiplexed flow cytometry:

  • Permeabilize cells for intracellular ESYT2 staining

  • Use fluorescently-conjugated antibodies or primary+secondary combinations

  • Include isotype controls for background subtraction

  • Measure mean fluorescence intensity (MFI) as quantitative readout

  • Combine with other markers for cell-type specific analysis

• Quantitative immunofluorescence microscopy:

  • Include calibration standards in each experiment

  • Use identical acquisition settings across samples

  • Apply automated image analysis algorithms to quantify:

    • Mean ESYT2 intensity per cell

    • ESYT2 puncta number and intensity

    • Co-localization coefficients with organelle markers

  • Employ single-molecule localization techniques for absolute quantification

• Capillary Western (Simple Western) analysis:

  • Automated size-based separation and immunodetection

  • High reproducibility and sensitivity for ESYT2 quantification

  • Small sample requirements (as little as 3 μg total protein)

  • Direct digital data output with reduced operator variability

• Proximity extension assay (PEA):

  • Highly sensitive method for protein quantification

  • Requires two antibodies recognizing different ESYT2 epitopes

  • Suitable for detecting low abundance ESYT2 in complex samples

  • Offers potential for multiplexing with other protein targets

For accurate ESYT2 quantification:

• Always include positive control samples with known ESYT2 expression (e.g., HeLa, K-562, LNCaP cells)

• Run standard curves with each experiment when possible

• Document antibody lot numbers and validate new lots against previous standards

• Consider the impact of sample preparation methods on quantitative measurements

These advanced quantification approaches provide more reliable measurements of ESYT2 expression levels, enabling meaningful comparisons across experimental conditions, cell types, or disease states .