EPSIN2 Antibody

What is the biological function of Epsin 2 (EPN2) in cellular processes?

Epsin 2 (EPN2) plays a crucial role in clathrin-mediated endocytosis where it aids in intracellular trafficking by binding to phospholipids . As a member of the epsin family of endocytic adaptors, it contains a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that facilitate protein-protein interactions . Research has demonstrated that epsins are integral to cell polarity-dependent processes, including cell migration and invasion through basement membranes . The ENTH domain functions as a signaling module by binding and inactivating Cdc42 GAPs, while the C-terminus interacts with other elements of the endocytic machinery such as AP2, EH-domain containing proteins, clathrin, and ubiquitylated cargo . This bi-modular architecture allows epsin to coordinate endocytosis with Cdc42-dependent signaling spatially and temporally .

Which applications are most suitable for EPSIN2 antibodies in research settings?

EPSIN2 antibodies are versatile tools applicable to multiple experimental techniques in research settings. Based on available research-grade antibodies, the most common and validated applications include:

• Western Blotting (WB): For protein expression quantification and molecular weight confirmation

• Immunohistochemistry (IHC): Including paraffin-embedded sections (IHC-P) for tissue localization

• Immunofluorescence (IF): For subcellular localization studies

• Flow Cytometry (FACS): For quantitative analysis in cell populations

• Enzyme-Linked Immunosorbent Assay (ELISA): For protein detection and quantification

When selecting an EPSIN2 antibody for your research, consider the validated applications, species reactivity, and clonality (monoclonal vs. polyclonal) based on your experimental design requirements .

How do I optimize Western blot protocols for detecting EPSIN2 in different tissue samples?

Optimizing Western blot protocols for EPSIN2 detection requires careful consideration of several parameters:

• Sample Preparation: Different tissues require different lysis buffers. For EPSIN2 detection, use a lysis buffer containing 1% NP-40 or Triton X-100 with protease inhibitors to preserve the protein's integrity.

• Protein Loading: Load 20-30 μg of total protein per lane. The predicted band size for human EPSIN2 is approximately 68 kDa .

• Antibody Dilution: Start with a 1:500 dilution for primary antibody as demonstrated in successful experiments with HEK-293T and NIH/3T3 cell lysates . Adjust based on signal strength.

• Incubation Time: Overnight incubation at 4°C typically yields optimal results.

• Detection Method: ECL (Enhanced Chemiluminescence) technique has been successfully employed for EPSIN2 detection .

• Controls: Include positive controls such as HEK-293T or NIH/3T3 whole cell lysates which have demonstrated clear EPSIN2 expression .

When troubleshooting, consider that EPSIN2 may undergo post-translational modifications in different tissues, potentially resulting in slight variations in molecular weight.

How can EPSIN2 antibodies be used to investigate cancer cell invasion and migration mechanisms?

EPSIN2 antibodies are valuable tools for investigating cancer cell invasion and migration mechanisms due to the protein's established role in these processes. Research has demonstrated that epsins are required for cell polarity-dependent processes of cell migration and invasion, with overexpression enhancing fibrosarcoma migration and invasion through basement membrane .

Methodological approach for such investigations:

• Knockdown-Rescue Experiments: Use siRNA to knockdown endogenous EPSIN2, followed by transfection with siRNA-resistant constructs and subsequent detection with EPSIN2 antibodies to verify expression levels .

• Immunofluorescence Analysis: Utilize EPSIN2 antibodies (dilution 1:100) to monitor subcellular localization during migration/invasion processes, paying particular attention to cell polarization markers .

• Co-localization Studies: Combine EPSIN2 antibodies with markers for RalBP1, Arf6, and Rac1 to investigate the signaling pathway interactions during invasion .

• Invasion Assays: Quantify invasion through basement membrane-coated filters following EPSIN2 manipulation, then use antibodies to verify protein levels in correlation with invasive capacity .

• GTPase Activation Assays: After seeding cells on fibronectin, use EPSIN2 antibodies alongside assays for Arf6 and Rac1 activation to correlate EPSIN2 levels with downstream signaling events .

This EPSIN2 migration/invasion pathway appears to involve Arf6 and Rac1 activation rather than receptor internalization, suggesting a novel mechanism through which upregulation of epsins in certain cancers may contribute to their invasive characteristics .

What are the considerations for studying EPSIN2 interactions with RalBP1 using immunoprecipitation?

Studying EPSIN2 interactions with RalBP1 requires careful optimization of immunoprecipitation protocols, as this interaction is critical in understanding EPSIN2's role in cell signaling and migration.

Key methodological considerations:

• Antibody Selection: Choose an EPSIN2 antibody validated for immunoprecipitation applications. Monoclonal antibodies like the 1C2 clone may provide more specific results .

• Buffer Optimization: Since the interaction between EPSIN2's ENTH domain and RalBP1 is enhanced by PtdIns(4,5)P₂ , consider including PtdIns(4,5)P₂ in your lysis and wash buffers to preserve physiologically relevant interactions.

• Control Experiments:

  • Use HT1080 or NIH3T3 cell lysates as positive controls, as endogenous EPSIN2-RalBP1 interactions have been successfully immunoprecipitated from these cells .

  • Include an isotype control antibody (IgG1 for mouse monoclonal antibodies) .

• Confirmation Methods: Verify direct interactions using complementary approaches:

  • GST pull-down assays with purified GST-RalBP1 and EPSIN2 ENTH domain

  • Surface Plasmon Resonance (SPR) to determine binding affinity (reported Kd in the μM range)

  • Analytical ultracentrifugation for further verification

• Subcellular Localization: Since this interaction preferentially occurs at the plasma membrane where both proteins and PtdIns(4,5)P₂ are enriched , consider membrane fractionation before immunoprecipitation to enrich for relevant complexes.

This interaction represents a novel pathway through which EPSIN2 contributes to cell migration and invasion, independent of its classic endocytic functions .

How do yeast and mammalian EPSIN2 functions compare, and what antibody considerations apply for comparative studies?

Comparing yeast and mammalian EPSIN2 functions requires understanding their evolutionary relationship and functional conservation while addressing specific antibody selection considerations.

Functional Comparison:

Antibody Considerations for Comparative Studies:

• Cross-reactivity: Most commercial antibodies are species-specific. For yeast studies, select antibodies specifically raised against yeast Ent2 .

• Domain Targeting: For comparative studies, select antibodies targeting conserved regions, particularly within the ENTH domain which maintains higher evolutionary conservation .

• Functional Analysis: While directly comparing proteins across species, consider functional complementation experiments:

  • Express mammalian EPSIN2 in yeast ent2Δ mutants

  • Use antibodies to verify expression and localization

  • Assess rescue of septation/division phenotypes

• Epitope Accessibility: The ENTH domain undergoes conformational changes upon PtdIns(4,5)P₂ binding , potentially affecting antibody recognition. Consider using antibodies targeting different regions when studying membrane-bound versus cytosolic protein pools.

Both yeast and mammalian epsins appear to function as bi-modular proteins where the ENTH domain serves as a signaling module while the C-terminus engages with the endocytic machinery . This conservation suggests fundamental importance in coordinating membrane trafficking with cellular signaling across eukaryotes.

What are common pitfalls when using EPSIN2 antibodies, and how can they be addressed?

Researchers frequently encounter several challenges when working with EPSIN2 antibodies. Understanding these pitfalls and implementing appropriate solutions can significantly improve experimental outcomes.

Common Pitfalls and Solutions:

• Cross-reactivity with EPSIN1:

  • Problem: EPSIN1 and EPSIN2 share structural similarities, particularly in the ENTH domain .

  • Solution: Select antibodies specifically validated against EPSIN2, particularly those targeting unique regions. Use EPSIN1 knockout/knockdown samples as controls to verify specificity .

• Variable Detection across Applications:

  • Problem: An antibody performing well in Western blot may not work for immunofluorescence.

  • Solution: Choose antibodies validated for your specific application. For EPSIN2, antibodies with proven multi-application performance (WB, IHC, IF) provide greater experimental flexibility .

• Inconsistent Results in Different Cell Types:

  • Problem: EPSIN2 expression levels and post-translational modifications vary across cell types.

  • Solution: Validate antibody performance in your specific cell type before proceeding with complex experiments. Determine optimal antibody concentrations for each cell type (e.g., 1:500 for Western blotting with HEK-293T and NIH/3T3 cell lysates) .

• Poor Signal in Immunofluorescence:

  • Problem: EPSIN2 may be present at low abundance or in specific subcellular locations.

  • Solution: Use a 1:100 dilution for immunofluorescence applications as validated in previous studies . Consider signal amplification methods or confocal microscopy for detecting subtle localization patterns.

• Inconsistent Immunoprecipitation Results:

  • Problem: EPSIN2 interactions may be transient or dependent on specific conditions.

  • Solution: Include PtdIns(4,5)P₂ in your buffers as this enhances EPSIN2 interactions with binding partners like RalBP1 . Cross-link protein complexes before immunoprecipitation for transient interactions.

Careful antibody selection, validation in your specific experimental system, and optimization of protocols are essential for successful EPSIN2 antibody applications.

How can researchers distinguish between EPSIN2 isoforms in experimental systems?

Distinguishing between EPSIN2 isoforms presents a significant challenge in experimental systems. This question addresses methodological approaches to differentiate between variant forms of this important endocytic adaptor protein.

Methodological Approaches:

• Antibody Selection for Isoform Specificity:

  • Select antibodies raised against unique regions that differentiate between EPSIN2 isoforms

  • For N-terminal variants, consider antibodies targeting amino acids 6-36 or other N-terminal regions

  • For C-terminal variants, antibodies targeting regions such as AA 431-480 may be more appropriate

• Western Blot Optimization:

  • Use high-resolution SDS-PAGE (8-10% gels) run for extended periods to resolve small molecular weight differences between isoforms

  • Consider using Phos-tag™ acrylamide gels to separate phosphorylated isoforms

  • The predicted band size for full-length human EPSIN2 is approximately 68 kDa, with isoforms potentially appearing at different molecular weights

• RT-PCR and qPCR Analysis:

  • Design primers that specifically amplify different EPSIN2 transcript variants

  • Combine with Western blot analysis using isoform-specific antibodies to correlate transcript and protein expression

• Functional Validation:

  • Use siRNA targeting specific isoforms followed by rescue experiments with siRNA-resistant constructs of individual isoforms

  • Monitor phenotypic outcomes in cell migration, invasion, or endocytosis assays to determine isoform-specific functions

• Subcellular Localization Analysis:

  • Different EPSIN2 isoforms may exhibit distinct subcellular localization patterns

  • Use immunofluorescence with isoform-specific antibodies (1:100 dilution) combined with markers for plasma membrane, endocytic structures, or other cellular compartments

This methodological framework allows researchers to comprehensively distinguish between EPSIN2 isoforms, enabling more precise characterization of their specific roles in cellular processes.

What controls should be included when validating EPSIN2 antibody specificity?

Proper validation of EPSIN2 antibody specificity is crucial for generating reliable and reproducible research results. This question outlines essential controls and validation steps researchers should implement.

Essential Controls for EPSIN2 Antibody Validation:

• Positive Controls:

  • Cell lines with confirmed EPSIN2 expression:

    • HEK-293T (human embryonic kidney cells)

    • NIH/3T3 (mouse embryo fibroblasts)

  • Tissue samples with known EPSIN2 expression:

    • Human brain cortex (validated for IHC at 1:100 dilution)

    • Tissues with high endocytic activity

• Negative Controls:

  • EPSIN2 knockout or knockdown samples:

    • siRNA-treated cells with verified EPSIN2 depletion

    • CRISPR/Cas9-generated EPSIN2 knockout cell lines

  • Primary antibody omission control

  • Isotype control (e.g., IgG1 for mouse monoclonal antibodies)

• Specificity Controls:

  • Pre-adsorption with immunizing peptide

  • Parallel testing with multiple EPSIN2 antibodies targeting different epitopes:

    • N-terminal (AA 6-36)

    • Internal regions (AA 120-300)

    • C-terminal (AA 431-480)

  • Cross-reactivity assessment with related proteins (especially EPSIN1)

• Application-Specific Controls:

  • For Western blot: Molecular weight ladder to confirm the expected 68 kDa band size

  • For IHC/IF: Subcellular localization consistent with endocytic adaptor function

  • For IP: Non-specific binding assessment using beads-only control

• Cross-Species Validation:

  • When using antibodies across species, validate specificity in each species:

    • Human, mouse, and rat samples for antibodies claiming multi-species reactivity

    • Monkey samples for antibodies with primate cross-reactivity

Implementation of these comprehensive controls ensures that experimental observations truly reflect EPSIN2 biology rather than artifacts of non-specific antibody binding or cross-reactivity with related proteins.

How can EPSIN2 antibodies be used to investigate its role in cancer progression and metastasis?

EPSIN2 antibodies offer valuable tools for investigating this protein's emerging role in cancer progression and metastasis. Research has established connections between epsin family members and invasive cancer characteristics, making this an important area for further study.

Methodological Approaches:

• Expression Analysis in Patient Samples:

  • Use EPSIN2 antibodies for immunohistochemistry (IHC-P, 1:100 dilution) on cancer tissue microarrays

  • Compare EPSIN2 expression between primary tumors and metastatic lesions

  • Correlate expression levels with clinical outcomes and metastatic potential

• Mechanistic Studies in Cancer Cell Lines:

  • Implement knockdown-rescue experiments using siRNA against EPSIN2 followed by transfection with siRNA-resistant constructs

  • Quantify effects on:

    • Cell migration (wound healing assays)

    • Invasion through basement membrane-coated filters (20-hour assay)

    • Activation of Arf6 and Rac1 GTPases (pull-down assays)

• Interaction Studies:

  • Use co-immunoprecipitation with EPSIN2 antibodies to identify cancer-specific interaction partners

  • Particularly focus on RalBP1 interactions, as this protein-protein interaction has been implicated in migration/invasion

  • Investigate how these interactions differ between normal and cancer cells

• Functional Imaging:

  • Employ fluorescently-tagged EPSIN2 antibodies (if available) or GFP-EPSIN2 constructs

  • Perform live-cell imaging to monitor EPSIN2 dynamics during cancer cell invasion

  • Co-localize with markers of invadopodia and other invasion-associated structures

• Therapeutic Target Validation:

  • Use EPSIN2 antibodies to monitor protein levels following treatment with potential therapeutic agents

  • Assess whether EPSIN2 inhibition or downregulation affects cancer cell survival, migration, or invasion

Research has demonstrated that overexpression of epsins enhances fibrosarcoma migration and invasion through basement membrane, suggesting that the observed up-regulation of either epsins or RalBP1 in certain cancers contributes to their invasive characteristics . This makes EPSIN2 a potentially important biomarker and therapeutic target worthy of in-depth investigation.

What is the relationship between EPSIN2 and septin assembly in cell division, and how can researchers study this?

The relationship between EPSIN2 and septin assembly in cell division represents an emerging area of research that bridges endocytosis with cytokinesis regulation. Studying this relationship requires specialized approaches focusing on the ENTH domain's interactions with septin regulatory proteins.

Relationship Overview:

Research in yeast has revealed that the ENTH domain of Epsin 2 (Ent2) plays a signaling role during cell division by interacting with septin assembly pathways . Overexpression of the ENTH domain of Ent2 (ENTH2) promotes the formation of chains of cells and aberrant septa, indicating a dominant-negative effect resulting from ENTH2-mediated interference with septin assembly . This suggests a previously unrecognized role for EPSIN2 in coordinating membrane trafficking with cell division.

Methodological Approaches for Study:

• ENTH Domain Overexpression Studies:

  • Express ENTH2 domain from a controllable promoter (e.g., methionine-repressible MET25 promoter)

  • Assess cell morphology, focusing on:

    • Formation of cell chains

    • Abnormal septum deposition

    • Viability using vital dyes like Methylene Blue

  • Electron microscopy to detect abnormal septa and cytosol entrapments (lacunae)

• Protein-Protein Interaction Analysis:

  • Map ENTH2 determinants responsible for septin phenotypes

  • Use co-immunoprecipitation with EPSIN2 antibodies to detect interactions with septin regulatory proteins (like Bem3 in yeast)

  • Confirm direct binding using purified proteins in GST pull-down assays

• Live Cell Imaging:

  • Visualize EPSIN2 and septin dynamics during cell division using fluorescently tagged proteins

  • Perform time-lapse microscopy to determine the temporal relationship between EPSIN2 localization and septin ring assembly

• Cross-Species Comparisons:

  • Determine whether mammalian EPSIN2 performs similar functions in septin regulation

  • Use EPSIN2 antibodies to examine co-localization with mammalian septins during cytokinesis

  • Assess whether EPSIN2 depletion in mammalian cells leads to septin disorganization and cytokinesis defects

• Phospholipid Dependency Analysis:

  • Investigate whether PtdIns(4,5)P₂ binding affects EPSIN2's interactions with septin regulatory proteins

  • This is particularly relevant as PtdIns(4,5)P₂ binding leads to conformational changes in the ENTH domain and enhances its interactions with binding partners

Understanding this relationship provides insight into how membrane trafficking proteins like EPSIN2 may coordinate endocytosis with cell division, potentially revealing new therapeutic targets for diseases involving dysregulated cell division.

What emerging techniques are enhancing EPSIN2 antibody applications in research?

Several cutting-edge techniques are expanding the utility of EPSIN2 antibodies in research, enabling more sophisticated investigations into this protein's multifaceted roles in cellular processes.

Emerging Techniques:

• Proximity Labeling Combined with Mass Spectrometry:

  • BioID or APEX2 fusion proteins with EPSIN2 can identify proximity interactors in living cells

  • When combined with EPSIN2 antibodies for validation, this approach can map the dynamic EPSIN2 interactome during processes like cell migration or division

  • Particularly valuable for identifying transient or context-specific interactions beyond established partners like RalBP1

• Super-Resolution Microscopy:

  • STORM, PALM, or STED microscopy using EPSIN2 antibodies enables visualization of protein localization at nanometer resolution

  • Can resolve EPSIN2 distribution within clathrin-coated pits and other endocytic structures

  • Allows co-localization studies with interaction partners with unprecedented spatial precision

• Live-Cell Antibody Fragments:

  • Single-chain variable fragments (scFvs) derived from EPSIN2 antibodies

  • When expressed as intrabodies or nanobodies, enable visualization or perturbation of EPSIN2 in living cells

  • Can be used to track endogenous EPSIN2 dynamics during cell division or migration

• CRISPR-Based Approaches:

  • CRISPR activation/inhibition systems to modulate EPSIN2 expression

  • CRISPR base editing or prime editing for introducing specific mutations

  • When combined with EPSIN2 antibodies for validation, enables precise dissection of domain-specific functions

• Tissue Clearing and 3D Imaging:

  • Techniques like CLARITY, iDISCO, or CUBIC combined with EPSIN2 antibodies

  • Enables whole-tissue or even whole-organ mapping of EPSIN2 expression patterns

  • Particularly valuable for understanding EPSIN2 distribution in complex tissues like brain

These emerging techniques, when combined with well-validated EPSIN2 antibodies, promise to significantly advance our understanding of this protein's roles in endocytosis, cell migration, division, and potentially in disease processes like cancer progression.

How might EPSIN2 antibodies contribute to therapeutic development targeting endocytic pathways?

EPSIN2 antibodies hold significant potential for contributing to therapeutic development targeting endocytic pathways, particularly in conditions where these pathways are dysregulated.

Potential Therapeutic Applications:

• Cancer Therapy Development:

  • EPSIN2 antibodies can help validate this protein as a therapeutic target based on its role in cancer cell migration and invasion

  • High-throughput screening for EPSIN2 inhibitors can use antibodies in target engagement assays

  • Patient stratification based on EPSIN2 expression levels (detected via IHC with antibodies) could identify those most likely to benefit from endocytosis-targeting therapies

• Antibody-Drug Conjugates:

  • If EPSIN2 undergoes internalization, therapeutic antibodies directed against accessible epitopes could potentially deliver cytotoxic payloads specifically to cells overexpressing EPSIN2

  • Particularly relevant for cancers where EPSIN2 is upregulated and contributes to invasive characteristics

• Modulation of Receptor Trafficking:

  • EPSIN2 antibodies can help map the role of this protein in trafficking of specific receptors

  • This knowledge could guide development of therapeutics that selectively modify receptor internalization or recycling

  • Particularly valuable for receptors that drive disease progression when dysregulated

• Diagnostic Development:

  • EPSIN2 antibodies could serve as diagnostic tools for conditions characterized by altered endocytic pathway function

  • Immunohistochemistry protocols (1:100 dilution for EPSIN2 antibodies) could be standardized for clinical use

  • Correlation with disease progression could establish EPSIN2 as a prognostic biomarker

• Blood-Brain Barrier Targeting:

  • Given EPSIN2 expression in brain cortex , understanding its role in blood-brain barrier endothelial cells could inform development of strategies for enhanced CNS drug delivery

  • Antibodies are essential tools for mapping EPSIN2 expression and function in these specialized endothelial cells