EMP2 Antibody, FITC conjugated

What is EMP2 and why is it a significant research target?

EMP2 (Epithelial Membrane Protein 2) functions as a key regulator of cell membrane composition by regulating protein surface expression. It plays critical roles in multiple cellular processes including cell migration, proliferation, contraction, and adhesion . EMP2 has emerged as a significant research target due to its upregulation in various cancers, particularly invasive breast cancer and triple-negative breast cancer (TNBC) . Additionally, EMP2 regulates transepithelial migration of neutrophils and negatively regulates caveolae formation by reducing CAV1 expression and increasing its lysosomal degradation . These diverse functions make EMP2 an important molecule for understanding cellular physiology and disease mechanisms.

How does FITC conjugation affect antibody functionality compared to unconjugated antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct visualization capabilities while potentially influencing antibody characteristics in the following ways:

• Binding affinity: Conjugation may slightly reduce binding affinity compared to unconjugated antibodies due to potential steric hindrance at the antigen-binding site, especially if conjugation occurs near this region

• Sensitivity: FITC has a quantum yield of approximately 0.9 and an excitation maximum of 495nm, making it adequately sensitive for most research applications, though less photostable than newer fluorophores

• Background signal: FITC exhibits pH sensitivity, with optimal fluorescence at pH >7.0, necessitating appropriate buffering systems in experimental designs

• Multiplexing capability: FITC's emission spectrum (519nm) may overlap with other fluorophores in multi-parameter experiments, requiring appropriate compensation controls

When using FITC-conjugated anti-EMP2 antibodies, researchers should conduct validation experiments comparing signal intensity and specificity against unconjugated primary antibody plus FITC-secondary antibody systems to ensure optimal performance in their specific applications.

What are the most validated applications for EMP2 antibody, FITC conjugated?

Based on research literature, FITC-conjugated EMP2 antibodies have been successfully employed in:

• Flow cytometry: For detection of cell surface EMP2 expression, as demonstrated in studies evaluating EMP2 expression on cancer cell lines like MDA-MB-231 and 4T1

• Immunofluorescence microscopy: For localization studies examining EMP2 co-localization with focal adhesion kinase (FAK)

• Live-cell imaging: For tracking EMP2 trafficking and internalization in real-time

• Immunohistochemistry: For detection of EMP2 in tissue sections, though this typically requires optimization of fixation methods

In validation studies, FITC-conjugated anti-EMP2 antibodies have demonstrated the ability to recognize both murine EMP2 (on 4T1 cells) and human EMP2 (on MDA-MB-231 cells), confirming cross-species reactivity for certain antibody clones .

How can EMP2 antibodies be utilized in breast cancer research models?

EMP2 antibodies serve multiple functions in breast cancer research:

• Detection and quantification: FITC-conjugated EMP2 antibodies can identify and quantify EMP2 expression levels across different breast cancer subtypes. Research has shown that EMP2 mRNA is upregulated in breast cancers, making it a potential biomarker

• Therapeutic targeting assessment: Studies have used anti-EMP2 antibodies to evaluate the efficacy of EMP2-targeted therapies. For example, anti-EMP2 IgG1 was found to bind to EMP2 on triple-negative breast cancer cell lines with an EC50 of 10.8 ng/mL

• Signaling pathway investigation: EMP2 antibodies can help investigate downstream signaling pathways affected by EMP2. Research has shown that treating MDA-MB-468 cells with anti-EMP2 IgG1 affects FAK and Src signaling pathways, which are crucial for cancer cell survival and metastasis

• Syngeneic mouse models: Anti-EMP2 therapies such as GrB-Fc-KS49 have demonstrated efficacy in TNBC syngeneic (4T1/FLuc) mouse models, reducing tumor volume, cell proliferation, and increasing cell death compared to controls

When designing experiments, researchers should include appropriate controls and consider using multiple detection methods to validate findings.

What role does EMP2 play in chlamydial infections and how can EMP2 antibodies aid research in this area?

EMP2 has been identified as a critical host protein in chlamydial infection pathogenesis:

• Infection mechanism: EMP2 appears to facilitate early chlamydial infection processes. Blockade of EMP2 using anti-EMP2 diabody (KS83) significantly reduces bacterial load in murine infection models

• Inflammatory response regulation: EMP2 blockade decreases production of proinflammatory cytokines (IFN-γ, TNF-α, GM-CSF) during early Chlamydia infection, suggesting EMP2's role in modulating host immune responses

• Research applications: FITC-conjugated EMP2 antibodies enable:

  • Visualization of EMP2 during infection progression

  • Assessment of co-localization with bacterial inclusion bodies

  • Quantification of EMP2 expression changes during infection cycles

• Therapeutic potential: Anti-EMP2 diabody treatment significantly reduced bacterial load, tissue production of inflammatory cytokines, recruitment of polymorphonuclear leukocytes, and local tissue inflammation in chlamydial infection models

Importantly, studies using anti-EMP2 diabody treatment demonstrated protection against chlamydial infection was associated with reduced tissue-associated cytokines and decreased recruitment of polymorphonuclear neutrophils (PMNs) throughout the genital tract, suggesting therapeutic potential for genital chlamydial infections .

What are the optimal staining protocols for detecting EMP2 using FITC-conjugated antibodies in flow cytometry?

Optimized Flow Cytometry Protocol for EMP2 Detection:

• Cell preparation:

  • Harvest cells using enzyme-free dissociation methods (e.g., EDTA) to preserve surface EMP2

  • Maintain viability >95% (confirm with viability dye)

  • Prepare 1×10^6 cells per sample in cold PBS with 2% FBS

• Blocking and staining:

  • Block with 5% normal serum from the same species as secondary antibody (if using indirect staining) for 20 minutes at 4°C

  • For direct staining: Apply FITC-conjugated anti-EMP2 antibody at optimized concentration (typically 1-10 μg/mL) for 30 minutes at 4°C in the dark

  • Include appropriate controls: isotype control, unstained, single-color controls for compensation

• Optimization considerations:

  • EMP2 detection may require signal amplification in cells with low expression

  • N-linked glycans may affect epitope recognition; consider PNgase treatment for certain applications

  • Permeabilization may be necessary for total EMP2 detection as opposed to surface-only detection

• Analysis parameters:

  • Analyze on flow cytometer with 488nm laser

  • Collect minimum 10,000 events per sample

  • Use hierarchical gating strategy: FSC/SSC → single cells → viable cells → EMP2+ population

This protocol has been validated for detecting both murine EMP2 on 4T1 cells and human EMP2 on MDA-MB-231 cells with high specificity .

How should co-localization studies of EMP2 with FAK be designed and analyzed?

Protocol for EMP2-FAK Co-localization Studies:

• Sample preparation:

  • Culture cells on fibronectin/collagen-coated coverslips (10 μg/mL) for 24-48 hours

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

• Immunostaining approach:

  • Block with 3% BSA for 1 hour

  • Primary staining options:

    • Option A: FITC-conjugated anti-EMP2 antibody + anti-phospho-FAK (Y576/577) primary antibody

    • Option B: Anti-EMP2 primary + FITC-conjugated secondary + anti-phospho-FAK (Y576/577) primary

  • Secondary staining: Use spectrally distinct fluorophore (e.g., Alexa Fluor 594) for FAK detection

  • Counterstain nuclei with DAPI

• Image acquisition and analysis:

  • Capture images using confocal microscopy with appropriate filter sets

  • Analyze co-localization quantitatively using:

    • Pearson's correlation coefficient (values >0.5 suggest meaningful co-localization)

    • Manders' overlap coefficient

    • Object-based co-localization analysis for focal adhesion structures

• Controls and validation:

  • Include single-stained samples for spectral bleed-through correction

  • Validate findings with proximity ligation assay or co-immunoprecipitation

  • Consider super-resolution techniques for detailed focal adhesion analysis

Based on published research, EMP2 and FAK show significant co-localization at focal adhesions, with approximately 24-30% of total FAK physically associated with EMP2 in ARPE-19 cells .

What statistical approaches are most appropriate for analyzing differential EMP2 expression across tissue samples?

Statistical Framework for EMP2 Expression Analysis:

• Preprocessing considerations:

  • Normalize fluorescence intensity data to account for batch effects

  • Transform data if necessary to achieve normal distribution (log-transformation often required)

  • Assess outliers using Grubbs' test or similar methods

• Statistical tests based on experimental design:

  Experimental Comparison	Recommended Test	Sample Size Recommendation
  Two independent groups	Student's t-test or Mann-Whitney U (non-parametric)	Minimum n=5 per group
  Multiple independent groups	One-way ANOVA with post-hoc tests (Tukey's or Dunnett's)	Minimum n=5 per group
  Matched/paired samples	Paired t-test or Wilcoxon signed-rank test	Minimum n=5 pairs
  Correlation with clinical parameters	Pearson's or Spearman's correlation	Minimum n=20
  Survival analysis	Kaplan-Meier with log-rank test	Minimum n=30

• Advanced analytical approaches:

  • Consider multivariate analysis when examining EMP2 expression alongside other markers

  • Use hierarchical clustering to identify patterns in EMP2 expression across sample types

  • Apply machine learning approaches for predictive modeling (e.g., random forest, support vector machines)

• Reporting requirements:

  • Always include effect size alongside p-values

  • Report exact p-values rather than thresholds

  • Include 95% confidence intervals where appropriate

  • Create clear data visualization (box plots, violin plots) showing individual data points

When analyzing EMP2 expression in cancer samples, studies have demonstrated significant upregulation of EMP2 in breast cancer tissues compared to normal mammary tissue, with particularly high expression in triple-negative breast cancer subtypes .

How can researchers troubleshoot weak or non-specific signals when using FITC-conjugated EMP2 antibodies?

Systematic Troubleshooting Approach:

• Weak signal issues and solutions:

  • Problem: Low EMP2 expression

    • Solution: Try signal amplification methods (tyramide signal amplification or use of more sensitive detection systems)

  • Problem: Epitope masking

    • Solution: Optimize antigen retrieval methods; consider PNgase treatment as EMP2 contains N-linked glycans that may interfere with antibody binding

  • Problem: Photobleaching

    • Solution: Minimize exposure to light; use anti-fade mounting media; consider switching to more photostable fluorophores

• Non-specific binding remediation:

  • Increase blocking time/concentration (5% BSA or 10% serum)

  • Optimize antibody dilution through titration experiments

  • Include additional washing steps with 0.1% Tween-20

  • Pre-adsorb antibody with relevant tissues/cells

• Validation approaches:

  • Confirm specificity using EMP2 knockdown/knockout controls

  • Compare staining pattern with alternative EMP2 antibody clones

  • Verify correct EMP2 molecular weight by Western blot (note: PNgase treatment may be required to remove N-linked glycans)

• Fluorescence-specific considerations:

  • Check pH of buffers (FITC fluorescence is optimal at pH >7.0)

  • Evaluate for autofluorescence in the FITC channel (especially in certain tissues)

  • Consider spectral unmixing if multiple fluorophores are used

Research has shown that EMP2 antibody detection can be affected by glycosylation state, and appropriate controls should be employed when studying heavily glycosylated variants of EMP2 .

What are the considerations for developing therapeutic antibodies targeting EMP2?

Key Factors in Anti-EMP2 Therapeutic Development:

• Target validation evidence:

  • EMP2 is upregulated in invasive breast cancer, including triple-negative breast cancer

  • Blockade of EMP2 reduces chlamydial infectivity in vitro and in vivo

  • GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein, has shown high specificity and cytotoxicity towards EMP2-positive cancer cells

• Antibody format considerations:

  • Full-length antibodies: Provide longer half-life and potential effector functions

  • Diabodies: Smaller size enables better tissue penetration; anti-EMP2 diabody KS83 has shown efficacy in reducing chlamydial infection

  • Antibody-drug conjugates: Enable targeted delivery of cytotoxic agents

  • Fusion proteins: GrB-Fc-KS49 combines granzyme B with anti-EMP2 antibody for specific cell killing

• Pharmacokinetic/pharmacodynamic optimization:

  • GrB-Fc-KS49 demonstrated biexponential plasma clearance with initial t₁/₂α=18.4 hours and slower terminal clearance t₁/₂β=73.1 hours

  • Ex vivo stability testing at 37°C indicated half-life exceeding 96 hours for some constructs

• Cross-reactivity considerations:

  • Some anti-EMP2 antibodies demonstrate cross-reactivity between human and murine EMP2

  • Epitope selection should consider species conservation for translational research

The development of GrB-Fc-KS49, which combines granzyme B with an anti-EMP2 single-chain antibody tethered through an IgG Fc domain, represents an innovative approach to specifically deliver a cytotoxic payload to EMP2-expressing cancer cells .

How can researchers investigate the role of EMP2 in coordinating FAK signaling and focal adhesion dynamics?

Methodological Approaches for EMP2-FAK Signaling Research:

• Quantitative phosphorylation analysis:

  • Western blot analysis using phospho-specific antibodies targeting different FAK phosphorylation sites (Y576/577, Y397, Y925)

  • Phosphoproteomic analysis for comprehensive phosphorylation profiling

  • Kinetic analysis of phosphorylation events following adhesion to extracellular matrix proteins

• Protein-protein interaction investigation:

  • Co-immunoprecipitation protocols:

    • Lyse cells in buffer containing 1% Triton X-100, 0.5% NP-40, 150mM NaCl, 50mM Tris-HCl pH 7.6, 5mM EDTA, 1mM PMSF, and protease inhibitors

    • Immunoprecipitate with anti-EMP2 antibody

    • Analyze precipitates for FAK content by Western blotting

  • Proximity ligation assay for in situ detection of EMP2-FAK interactions

  • FRET-based approaches to measure dynamic interactions

• Functional consequence assessment:

  • Cell adhesion assays on various ECM substrates

  • Migration and invasion assays in 2D and 3D systems

  • Live-cell imaging of focal adhesion dynamics using fluorescently tagged proteins

• Genetic manipulation strategies:

  • EMP2 knockout/knockdown to assess FAK phosphorylation state

  • EMP2 overexpression systems (validated approach shows increased FAK phosphorylation at multiple sites)

  • Domain mapping using truncated EMP2 constructs to identify interaction regions

Research has demonstrated that approximately 24-30% of total FAK physically associates with EMP2 in ARPE-19 cells, and EMP2 overexpression leads to increased FAK phosphorylation at multiple sites. This physical association results in functional cellular alterations including increased focal adhesion density, changes in actin cytoskeleton organization, and increased cellular adhesive capacity .

What are emerging applications of EMP2 antibodies in novel therapeutic approaches?

Innovative Therapeutic Strategies:

• Immune modulation approaches:

  • EMP2 antibodies may modulate host immune responses during infection, as evidenced by reduced inflammatory cytokine production following anti-EMP2 diabody treatment in chlamydial infection models

  • Potential applications in reducing immunopathology associated with chronic infections

• Novel fusion protein designs:

  • GrB-Fc-KS49 represents a pioneering approach combining granzyme B with anti-EMP2 antibody

  • This construct showed:

    • Comparable GrB enzymatic activity to commercial GrB

    • High affinity binding to EMP2 peptide (dissociation constant in picomolar range)

    • Rapid internalization into EMP2+ cancer cells

    • In vitro cytotoxicity with IC₅₀ values below 100 nM for most positive cell lines

• Multi-targeting strategies:

  • Given EMP2's interaction with FAK, dual targeting of EMP2 and FAK signaling pathways may provide synergistic therapeutic effects

  • Combined blockade of EMP2 and integrin signaling represents an unexplored therapeutic avenue

• Theranostic applications:

  • Dual-function antibodies that combine imaging capabilities with therapeutic activity

  • EMP2 antibodies conjugated with imaging agents could enable real-time monitoring of therapy response

The development of GrB-Fc-KS49 has demonstrated promising preclinical efficacy against triple-negative breast cancer in syngeneic mouse models, reducing tumor volume and cell proliferation while increasing cell death compared to controls .

How might single-cell analysis technologies enhance our understanding of EMP2 expression heterogeneity?

Single-Cell Technologies for EMP2 Research:

• Single-cell RNA sequencing applications:

  • Reveals heterogeneity of EMP2 expression within seemingly homogeneous populations

  • Enables correlation of EMP2 expression with specific cell states or phenotypes

  • Can identify co-expression patterns with interacting partners (e.g., FAK, integrins)

• Mass cytometry (CyTOF) approaches:

  • Allows simultaneous detection of EMP2 alongside dozens of other proteins

  • Metal-tagged antibodies avoid fluorescence spillover issues

  • Can reveal rare cell populations with unique EMP2 expression patterns

• Spatial transcriptomics integration:

  • Combines single-cell resolution with spatial context

  • Particularly valuable for understanding EMP2 expression at tumor-stroma interfaces

  • Technologies like 10x Visium or Nanostring GeoMx provide spatial context to expression data

• Implementation considerations:

  • Sample preparation protocols must preserve EMP2 epitope integrity

  • Antibody validation for single-cell applications is essential

  • Computational analysis requires specialized pipelines for heterogeneity assessment

These emerging technologies will facilitate understanding of how EMP2 expression varies across different cell types within complex tissues and how this heterogeneity contributes to disease processes.

What are the current challenges in utilizing EMP2 as a biomarker for cancer diagnosis and prognosis?

Critical Challenges and Potential Solutions:

• Standardization issues:

  • Challenge: Lack of standardized cutoff values for "high" versus "low" EMP2 expression

  • Approach: Develop reference standards and consensus guidelines for EMP2 quantification across different detection platforms

• Technical considerations:

  • Challenge: Post-translational modifications (glycosylation) affect antibody binding and detection consistency

  • Approach: Development of antibodies targeting invariant epitopes or standardized deglycosylation protocols prior to analysis

• Biological complexity:

  • Challenge: Context-dependent functions of EMP2 across different cancer types

  • Approach: Multi-parametric analysis incorporating EMP2 with other markers to create cancer-specific signature profiles

• Clinical validation requirements:

  • Challenge: Limited large-scale clinical studies validating EMP2 as a prognostic/diagnostic marker

  • Approach: Design of retrospective and prospective studies with appropriate statistical power to validate clinical utility

• Detection sensitivity:

  • Challenge: Low abundance in certain sample types (e.g., liquid biopsies)

  • Approach: Development of amplification methods or highly sensitive detection platforms (digital ELISA, etc.)