EMP2 Antibody, HRP conjugated

What is EMP2 and why is it targeted in cancer research?

EMP2 is a tetraspan protein belonging to the GAS-3/PMP22 gene family. It has emerged as a significant therapeutic target due to its upregulation in multiple cancer types. EMP2 protein expression is minimal in normal tissues like mammary tissue but is significantly upregulated in 63% of invasive breast cancer tumors and 73% of triple negative breast cancer cases . Recent studies have demonstrated that EMP2 promotes tumor growth in several cancers including breast cancer, hepatocellular carcinoma (HCC), and glioblastoma (GBM) . The protein's significant tissue specificity and heterogeneity in various tumor tissues make it an excellent candidate for targeted therapies and diagnostic applications.

How are HRP-conjugated anti-EMP2 antibodies detected in experimental procedures?

In experimental procedures, HRP-conjugated anti-EMP2 antibodies are typically detected through colorimetric or chemiluminescent methods. For instance, in ELISA assays, bound antibodies are detected with HRP-conjugated secondary antibodies (such as goat anti-human IgG) followed by the addition of TMB (3,3',5,5'-Tetramethylbenzidine) solution, which produces a color change that can be measured at 450 nm using a microplate reader . In Western blot applications, HRP-conjugated secondary antibodies enable visualization of EMP2 expression using ECL (Enhanced Chemiluminescence) detection reagents . For optimal results, researchers typically use a 1:2000 dilution of primary anti-EMP2 antisera followed by appropriate HRP-conjugated secondary antibodies diluted according to manufacturer specifications.

What are the properties of EMP2 that make it suitable for antibody detection?

EMP2 possesses several properties that make it an excellent target for antibody detection:

• Membrane localization: As a tetraspan protein, EMP2 has multiple extracellular domains accessible to antibodies.

• Differential expression: EMP2 shows minimal expression in normal tissues but is significantly upregulated in multiple cancer types .

• Conserved domains: EMP2 contains conserved regions across human and murine species, allowing antibodies to recognize both human and mouse EMP2 .

• Stable expression: Once upregulated in cancer tissues, EMP2 shows consistent expression, making it a reliable biomarker.

• Functional significance: EMP2 actively participates in signaling pathways related to cancer progression, including FAK/Src signaling and the integrin pathway .

What controls should be included when working with HRP-conjugated anti-EMP2 antibodies?

When working with HRP-conjugated anti-EMP2 antibodies, the following controls are essential:

These controls help validate experimental results and troubleshoot potential issues with specificity and sensitivity.

How can EMP2 expression be quantitatively assessed using HRP-conjugated antibodies?

EMP2 expression can be quantitatively assessed using several methods with HRP-conjugated antibodies:

• Western Blot Densitometry: After detection with HRP-conjugated antibodies and ECL visualization, band intensities can be quantified using densitometry software. Normalization to loading controls like β-actin is essential for accurate quantification .

• ELISA Quantification: A standard curve using recombinant EMP2 peptide can be established to determine absolute protein concentration in samples. This approach has demonstrated an EC50 of 10.8 ng/mL for anti-EMP2 IgG1 binding to human EMP2 peptide .

• Tissue Microarray Analysis: In clinical samples, the H-score method provides a semi-quantitative assessment of EMP2 expression. The formula [3(%a) + 2(%b) + 1(%c)]/100 is used, where a, b, and c represent the percentages of cells staining at intensities 3, 2, and 1, respectively .

• Flow Cytometry Mean Fluorescence Intensity: After staining with anti-EMP2 primary antibody and HRP-conjugated secondary antibody, the mean fluorescence intensity can be measured to quantify surface expression levels of EMP2 .

What are the optimal conditions for using HRP-conjugated anti-EMP2 antibodies in Western blotting?

For optimal Western blot detection of EMP2 using HRP-conjugated antibodies:

• Sample Preparation: Cell lysates should be prepared in RIPA buffer with protease and phosphatase inhibitors. For membrane proteins like EMP2, avoiding excessive heat during sample preparation preserves protein structure.

• Gel Electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation of EMP2 (approximately 20 kDa).

• Transfer Conditions: Transfer to PVDF membranes (preferred over nitrocellulose for membrane proteins) at 100V for 1 hour in cold transfer buffer containing 20% methanol.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Use rabbit anti-human EMP2 antisera at 1:2,000 dilution in blocking buffer overnight at 4°C .

• Secondary Antibody: Apply HRP-conjugated goat anti-rabbit antibody at manufacturer-recommended dilution (typically 1:5,000 to 1:10,000) for 1 hour at room temperature.

• Detection: Visualize using ECL detection reagents with exposure times optimized for signal-to-noise ratio.

• Controls: Include β-actin detection as a loading control using anti-β-actin primary antibody and appropriate HRP-conjugated secondary antibody .

How can HRP-conjugated anti-EMP2 antibodies be used to study EMP2-mediated signaling pathways?

HRP-conjugated anti-EMP2 antibodies are valuable tools for investigating EMP2-mediated signaling pathways through several approaches:

• Activation Status Analysis: After treatment with anti-EMP2 IgG1 or control IgG (100 μg/mL for 2 hours), cells can be plated to activate FAK and Src, then lysed after 12 hours. Western blotting with phospho-specific antibodies (anti-576/577p-FAK, anti-416 p-Src) alongside total protein antibodies reveals EMP2's influence on these signaling pathways .

• Temporal Signaling Dynamics: Time-course experiments monitoring phosphorylation changes after EMP2 activation or inhibition help elucidate signaling kinetics.

• Inhibitor Studies: Combining anti-EMP2 treatments with specific pathway inhibitors can reveal hierarchical relationships in signaling cascades.

• Co-immunoprecipitation: Using anti-EMP2 antibodies for immunoprecipitation followed by detection with HRP-conjugated antibodies against potential interacting partners can identify novel protein-protein interactions.

• Integrin Pathway Analysis: EMP2 enhances the invasive capacity of cancer cells by activating integrins, particularly αvβ3 integrin. HRP-conjugated antibodies can detect changes in integrin expression and activation status following EMP2 modulation .

What approaches can be used to validate the specificity of HRP-conjugated anti-EMP2 antibodies?

To validate the specificity of HRP-conjugated anti-EMP2 antibodies, researchers should implement these complementary approaches:

• Genetic Validation: Compare antibody binding between:

  • Wild-type cells and EMP2 knockdown/knockout cells

  • Cells with endogenous EMP2 levels versus those with EMP2 overexpression (e.g., HEC1a/V versus HEC1a/EMP2 cells with 2-4 fold increased EMP2 expression)

• Peptide Competition: Pre-incubate antibodies with excess EMP2-specific peptides before application to samples. Specific binding should be significantly reduced.

• Cross-Species Reactivity: Test antibody binding to both human and murine EMP2, as demonstrated in studies with anti-EMP2 IgG1 showing binding to both human (MDA-MB-231) and murine (4T1) cell lines .

• Negative Control Cell Lines: Verify absence of signal in EMP2-negative cell lines, such as human Ramos lymphoma cells .

• Multiple Detection Methods: Confirm consistent EMP2 detection across different methods (Western blot, ELISA, flow cytometry, immunohistochemistry).

• Antibody Characterization: Analyze antibody structure under reducing and non-reducing conditions to confirm expected molecular weights (~150 kDa for full antibody, ~60 kDa for heavy chain, and ~20 kDa for light chain) .

How can researchers optimize immunohistochemistry protocols using HRP-conjugated anti-EMP2 antibodies?

For optimal immunohistochemistry protocols with HRP-conjugated anti-EMP2 antibodies:

• Tissue Preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections cut at 4-5 μm thickness. Fresh frozen sections may provide better antigen preservation but require different fixation protocols.

• Antigen Retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes is typically effective for revealing EMP2 epitopes. For challenging samples, proteinase K treatment may be considered as an alternative.

• Endogenous Peroxidase Blocking: Quench endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 minutes.

• Background Reduction: Block non-specific binding with 5% normal serum from the same species as the secondary antibody for 1 hour at room temperature.

• Primary Antibody Optimization: Perform titration experiments with anti-EMP2 antibody (typically starting at 1:100-1:500) to determine optimal concentration.

• Detection System: Use an appropriate HRP-conjugated secondary antibody or polymer-based detection system for enhanced sensitivity.

• Signal Development: Develop with DAB (3,3'-diaminobenzidine) and optimize timing to avoid overdevelopment.

• Counterstaining: Use hematoxylin for nuclear counterstaining.

• Controls: Include positive controls (known EMP2-positive tissues), negative controls (EMP2-negative tissues), and technical controls (omitting primary antibody).

What are common troubleshooting strategies for experiments using HRP-conjugated anti-EMP2 antibodies?

When facing challenges with HRP-conjugated anti-EMP2 antibodies, consider these troubleshooting strategies:

How can HRP-conjugated anti-EMP2 antibodies be used to study the role of EMP2 in cancer progression?

HRP-conjugated anti-EMP2 antibodies offer multiple approaches to investigate EMP2's role in cancer progression:

• Expression Correlation Studies: Analyze EMP2 expression across cancer stages and correlate with clinical outcomes. In breast cancer, EMP2 is upregulated in 63% of invasive tumors and 73% of triple-negative cases, suggesting its role in aggressive disease .

• Signaling Pathway Analysis: Investigate how EMP2 modulates critical oncogenic pathways. Anti-EMP2 antibodies reveal that EMP2 activates FAK/Src signaling, promoting invasion and inhibiting apoptosis in breast cancer .

• Functional Assays: Combine anti-EMP2 treatments with cellular assays to assess:

  • Proliferation: Anti-EMP2 IgG1 inhibits proliferation in cancer cell lines

  • Invasion: EMP2 modulation affects invasive capacity through integrin activation

  • Apoptosis: Anti-EMP2 treatment promotes apoptosis in cancer cells

  • Autophagy: In hepatocellular carcinoma, EMP2 induces autophagy, affecting invasive capacity

• In Vivo Studies: Use anti-EMP2 antibodies in mouse models to track:

  • Tumor growth inhibition in xenograft models

  • Metastatic potential in syngeneic models

  • ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) responses

• Biomarker Validation: Evaluate EMP2 as a diagnostic or prognostic marker by correlating expression with clinical outcomes across patient cohorts.

What considerations are important when developing therapeutic anti-EMP2 antibodies with or without HRP conjugation?

When developing therapeutic anti-EMP2 antibodies, researchers should consider:

• Antibody Format: Fully human anti-EMP2 IgG1 antibodies have shown efficacy in preclinical studies, offering advantages over antibody fragments (diabodies) for therapeutic applications .

• Epitope Selection: Target conserved extracellular domains of EMP2 to ensure accessibility and functional effects. The anti-EMP2 IgG1 targeting the extracellular loop of human EMP2 showed an EC50 of 10.8 ng/mL .

• Cross-Species Reactivity: Develop antibodies recognizing both human and murine EMP2 to facilitate translational research, as demonstrated by anti-EMP2 IgG1 binding to both human cancer cells (MDA-MB-231) and murine mammary tumor cells (4T1) .

• Mechanism of Action: Consider multiple mechanisms:

  • Direct inhibition of EMP2-mediated signaling

  • Antibody-dependent cell-mediated cytotoxicity (ADCC)

  • Complement-dependent cytotoxicity (CDC)

  • Direct induction of apoptosis

• Target Validation: Confirm EMP2 expression across target cancer types. EMP2 is upregulated in multiple cancers including breast cancer (73% of triple-negative cases), hepatocellular carcinoma, and glioblastoma (95% of patients) .

• Safety Profile: Assess potential off-target effects by thoroughly evaluating EMP2 expression in normal tissues, which is typically minimal in tissues like normal brain and mammary tissue .

How does EMP2 expression correlate with cancer progression across different tumor types?

EMP2 expression demonstrates significant correlations with cancer progression across multiple tumor types:

• Breast Cancer: EMP2 is upregulated in 63% of invasive breast cancers and 73% of triple-negative breast cancers . Global gene signature studies have linked EMP2 mRNA upregulation with advanced disease and circulating breast tumor cells .

• Hepatocellular Carcinoma (HCC): Bioinformatic and immunohistochemical analyses reveal significant upregulation of EMP2 in HCC tissues, with expression increasing progressively from hepatitis to cirrhosis and ultimately to HCC .

• Glioblastoma (GBM): EMP2 has low or undetectable expression in normal brain but is highly expressed in GBM, with 95% of patients showing some level of expression . EMP2 expression significantly correlates with activated Src kinase in patient samples .

• Ovarian and Endometrial Cancers: EMP2 serves as a prognostic indicator in these cancers, with its expression correlating with poor survival and/or advanced disease .

The consistent upregulation of EMP2 across these diverse cancer types suggests a fundamental role in malignant transformation and progression, making it a promising pan-cancer therapeutic target.

What are the molecular mechanisms by which EMP2 promotes cancer cell invasion and metastasis?

EMP2 promotes cancer cell invasion and metastasis through several interconnected molecular mechanisms:

• Integrin Pathway Activation: EMP2 enhances the surface expression of specific integrins, particularly αvβ3 integrin in glioblastoma, which activates downstream signaling pathways promoting migration and invasion .

• FAK/Src Signaling Axis: EMP2 activates focal adhesion kinase (FAK) and Src kinases, as evidenced by increased phosphorylation of FAK at residues 576/577 and Src at residue 416 following EMP2 activation . This signaling axis is crucial for cellular adhesion, migration, and invasion.

• Autophagy Regulation: In hepatocellular carcinoma, EMP2 induces autophagy through a bidirectional regulatory mechanism that synergistically influences the invasive and metastatic potential of cancer cells .

• Anti-Apoptotic Effects: EMP2 inhibits apoptosis in cancer cells, contributing to tumor cell survival during the metastatic process .

• ECM Interaction Modulation: As a tetraspan protein, EMP2 likely organizes membrane microdomains that facilitate interactions with the extracellular matrix, promoting cell motility and invasion.

These mechanisms collectively create a pro-invasive phenotype that enhances the metastatic potential of cancer cells expressing elevated levels of EMP2.

What novel research directions are emerging for EMP2-targeted therapies beyond conventional antibody approaches?

Emerging research directions for EMP2-targeted therapies include:

• Antibody-Drug Conjugates (ADCs): Coupling anti-EMP2 antibodies with cytotoxic payloads could enhance their therapeutic efficacy while maintaining specificity.

• Bispecific Antibodies: Developing antibodies that simultaneously target EMP2 and other cancer-associated antigens or immune cell receptors could enhance therapeutic efficacy through multiple mechanisms.

• CAR-T Cell Therapy: Engineering T cells with chimeric antigen receptors targeting EMP2 represents a potential cellular immunotherapy approach, particularly for solid tumors expressing high levels of EMP2.

• Small Molecule Inhibitors: Identifying small molecules that disrupt EMP2 interactions with signaling partners (e.g., integrins or FAK/Src) could provide alternatives to antibody-based approaches.

• Gene Silencing Approaches: siRNA or CRISPR-based therapies targeting EMP2 could complement antibody approaches, particularly for cancers with intracellular EMP2 signaling dependencies.

• Combination Therapies: Integrating anti-EMP2 therapies with existing treatment modalities (e.g., chemotherapy, radiation, immune checkpoint inhibitors) could yield synergistic effects, as EMP2 modulates multiple oncogenic pathways.

• Biomarker-Driven Patient Selection: Developing companion diagnostics to identify patients with EMP2-dependent tumors would enhance the clinical applicability of targeted therapies.

What challenges remain in translating EMP2-targeted therapies from preclinical models to clinical applications?

Several significant challenges must be addressed to translate EMP2-targeted therapies to clinical applications:

• Heterogeneous Expression: While EMP2 is upregulated in many cancer tissues, expression levels vary between patients and even within different regions of the same tumor. Strategies for addressing this heterogeneity are needed for effective therapy.

• Therapeutic Window: Although EMP2 expression is minimal in most normal tissues, comprehensive safety studies are necessary to identify potential off-target effects in tissues with low but functionally important EMP2 expression.

• Resistance Mechanisms: Cancer cells may develop resistance to EMP2-targeted therapies through compensatory signaling pathways or downregulation of EMP2. Understanding and countering these mechanisms will be crucial for sustained clinical efficacy.

• Delivery Challenges: For solid tumors, particularly brain tumors like glioblastoma, ensuring sufficient antibody penetration across the blood-brain barrier and throughout the tumor mass remains challenging.

• Biomarker Development: Developing reliable biomarkers to predict response to anti-EMP2 therapy and monitor treatment efficacy will be essential for clinical implementation.

• Combination Strategies: Identifying optimal combination approaches that leverage EMP2 inhibition alongside standard-of-care treatments requires extensive preclinical and early-phase clinical studies.

• Manufacturing and Scalability: Producing consistent, high-quality antibodies or other EMP2-targeted therapeutics at scale presents technical and economic challenges.

Addressing these challenges will require collaborative efforts across basic research, translational medicine, and clinical development to realize the therapeutic potential of EMP2-targeted approaches.