EPSTI1 Antibody, Biotin conjugated

What is EPSTI1 and what are its primary biological functions?

EPSTI1 (Epithelial Stromal Interaction 1) is a protein that demonstrates tissue-specific expression patterns with significant implications in both normal physiology and disease states. Real-time PCR analysis reveals highest expression in the placenta, with strong expression also observed in the small intestine, spleen, salivary gland, and testes. Normal breast tissue and several other tissues show comparatively lower expression levels .

Functionally, EPSTI1 plays a crucial role in immune regulation, particularly in macrophage polarization. It is required for proper regulation of gene expression during M1 versus M2 macrophage differentiation. Additionally, EPSTI1 appears to be involved in RELA/p65 and STAT1 phosphorylation and nuclear localization upon macrophage activation . These functions position EPSTI1 as an important mediator in inflammatory responses and immune system modulation.

How does EPSTI1 expression vary across different pathological conditions?

EPSTI1 expression exhibits remarkable variability across different pathological conditions, particularly in cancer:

• In breast carcinomas, EPSTI1 is significantly upregulated (range 5.6-72.1) compared to normal breast tissue .

• In oral squamous cell carcinoma (OSCC), EPSTI1 is markedly upregulated across multiple cell lines (HSC2, HSC3, HSC3-M3, and HSC4) compared to control cell lines .

• Conversely, in lung squamous cell carcinoma (LSCC), EPSTI1 is significantly downregulated in multiple cell lines (LK-2, EBC-1, and H226) .

• In inflammatory conditions such as pneumonia models, EPSTI1 is highly expressed in LPS-treated cells, correlating with increased inflammatory responses .

This differential expression pattern suggests that EPSTI1 may serve contrasting functions depending on the specific pathological context, functioning as an oncogene in OSCC but potentially as a tumor suppressor in LSCC .

What applications are biotin-conjugated EPSTI1 antibodies most suitable for?

Biotin-conjugated EPSTI1 antibodies are particularly valuable for immunoassay techniques that leverage the high-affinity biotin-streptavidin interaction system. These applications include:

• Immunohistochemistry (IHC): Biotin-conjugated antibodies provide enhanced signal amplification when used with streptavidin-HRP detection systems, making them ideal for detecting EPSTI1 in tissue sections. This is particularly useful for analyzing EPSTI1 expression in breast cancer tissues where expression varies significantly from normal tissue .

• Flow Cytometry: The biotin-streptavidin system offers superior sensitivity for detecting low-abundance EPSTI1 in cell populations, especially when analyzing macrophage polarization states where EPSTI1 plays a regulatory role .

• ELISA: Biotin-conjugated EPSTI1 antibodies enable high-sensitivity detection in quantitative immunoassays. The antibody can be used in both direct and sandwich ELISA formats .

• Immunoprecipitation: The strong biotin-streptavidin interaction allows efficient pull-down of EPSTI1 and its interaction partners, facilitating the study of its role in signaling pathways such as NF-κB .

When selecting a biotin-conjugated EPSTI1 antibody, researchers should consider the specific epitope recognized by the antibody (e.g., AA 1-100) and the validated reactivity with species of interest (human, mouse, or pig) .

How can biotin-conjugated EPSTI1 antibodies be utilized to investigate its role in the NF-κB signaling pathway?

Biotin-conjugated EPSTI1 antibodies provide powerful tools for investigating EPSTI1's role in the NF-κB signaling pathway through multiple sophisticated approaches:

• Chromatin Immunoprecipitation (ChIP): Biotin-conjugated EPSTI1 antibodies can be used to isolate EPSTI1-bound chromatin complexes, followed by sequencing to identify genomic regions where EPSTI1 may regulate NF-κB-dependent gene expression. The biotin tag facilitates efficient pull-down using streptavidin magnetic beads without disturbing protein-DNA interactions.

• Co-immunoprecipitation with Proximity Labeling: When combined with BioID or APEX2 proximity labeling systems, biotin-conjugated EPSTI1 antibodies can help identify transient or weak interactions between EPSTI1 and NF-κB pathway components. Research has shown that EPSTI1 knockdown visibly decreases the ratios of p-p65/p65 and p-IκBα/IκBα induced by LPS stimulation .

• Multiplexed Imaging: Using differently colored streptavidin conjugates, biotin-conjugated EPSTI1 antibodies can be combined with antibodies against NF-κB components for advanced co-localization studies through confocal microscopy or super-resolution imaging. This approach can visualize the subcellular localization dynamics of EPSTI1 during NF-κB activation.

• Proximity Ligation Assay (PLA): Biotin-conjugated EPSTI1 antibodies paired with antibodies against p65, IκBα, or other NF-κB components can generate fluorescent signals only when proteins are in close proximity (<40 nm), providing spatial resolution for protein interactions in intact cells.

Studies have demonstrated that EPSTI1 knockdown inactivates the NF-κB pathway by reducing p-p65/p65 and p-IκBα/IκBα ratios, suggesting EPSTI1 positively regulates NF-κB signaling during inflammatory responses .

What methodological approaches can resolve the seemingly contradictory roles of EPSTI1 in different cancer types?

The contrasting functions of EPSTI1 in different cancers (oncogenic in OSCC but tumor-suppressive in LSCC) present an intriguing research question that can be addressed using biotin-conjugated EPSTI1 antibodies through the following methodological approaches:

• Cancer-Specific Interactome Analysis:

  • Conduct immunoprecipitation using biotin-conjugated EPSTI1 antibodies in both OSCC and LSCC cell lines

  • Analyze pulled-down protein complexes via mass spectrometry to identify cancer-specific binding partners

  • Compare interactomes to identify differential protein interactions that might explain the opposing functions

• Tissue-Specific Transcriptional Regulation:

  • Perform ChIP-seq using biotin-conjugated EPSTI1 antibodies in both cancer types

  • Identify differentially bound genomic regions and associated genes

  • Validate through reporter assays with tissue-specific promoters

• Isoform-Specific Expression Analysis:

  • Design experiments using biotin-conjugated antibodies targeting different EPSTI1 epitopes

  • Determine if different cancer types express distinct EPSTI1 isoforms with varying functions

  • Confirm through isoform-specific knockdown and overexpression studies

• Post-Translational Modification Profiling:

  • Immunoprecipitate EPSTI1 using biotin-conjugated antibodies from both cancer types

  • Analyze PTMs through mass spectrometry to identify cancer-specific modifications

  • Validate functional consequences through site-directed mutagenesis

This systematic approach addresses the dichotomy observed in EPSTI1 function, where knockdown in OSCC and overexpression in LSCC both suppressed cell proliferation, induced G1 phase cell-cycle arrest, upregulated p21, and downregulated CDK2 and cyclin D1 .

How can biotin-conjugated EPSTI1 antibodies be applied to study macrophage polarization in tumor microenvironments?

Biotin-conjugated EPSTI1 antibodies offer powerful methodological approaches for investigating EPSTI1's role in macrophage polarization within tumor microenvironments:

• Multiplex Immunohistochemistry Protocol:

  • Sample preparation: Fix tumor tissues with 4% paraformaldehyde followed by paraffin embedding

  • Deparaffinize sections and perform antigen retrieval in TE buffer pH 9.0

  • Apply biotin-conjugated EPSTI1 antibody (1:50-1:100 dilution) alongside markers for M1 (CD80, iNOS) and M2 (CD163, Arg1) macrophages

  • Develop using different streptavidin-fluorophore conjugates (for EPSTI1) and HRP-conjugated secondary antibodies with tyramide signal amplification (for other markers)

  • Analyze co-localization and expression patterns using multispectral imaging

• Flow Cytometry Analysis of TAMs:

  • Isolate cells from tumor tissues using enzymatic digestion

  • Perform surface staining for macrophage markers (CD11b, F4/80)

  • Fix and permeabilize cells for intracellular staining with biotin-conjugated EPSTI1 antibody

  • Analyze data using dimensionality reduction techniques (tSNE, UMAP) to identify EPSTI1-expressing macrophage subpopulations

• In vitro Polarization Studies:

  • Culture macrophages under M1 (LPS+IFNγ) or M2 (IL-4+IL-13) polarizing conditions

  • Track EPSTI1 expression kinetics using biotin-conjugated antibodies in flow cytometry or immunofluorescence

  • Perform EPSTI1 knockdown/overexpression experiments to assess impact on polarization markers

• Ex vivo Analysis of Patient Samples:

This methodological approach leverages EPSTI1's known involvement in M1 macrophage polarization and regulation of gene expression during M1 versus M2 macrophage differentiation , while investigating its differential roles across cancer types .

What is the optimal protocol for verifying the degree of biotinylation in EPSTI1 antibody conjugates?

An optimal protocol for verifying the degree of biotinylation in EPSTI1 antibody conjugates must overcome several technical challenges, including multivalency issues and steric hindrances. Based on advanced methodologies, here is a detailed protocol:

Materials Required:

• Biotin-conjugated EPSTI1 antibody sample

• Proteinase K

• Anti-biotin antibody

• Biotin standards (1-100 pmol)

• 96-well microtiter plate (high binding)

• PBS with 0.1% Tween-20 (PBST)

• 1% BSA in PBST

• Streptavidin-HRP conjugate

• TMB substrate

• 2M H₂SO₄ for stopping reaction

• Plate reader

Protocol Steps:

• Sample Preparation:

  • Dilute biotin-conjugated EPSTI1 antibody to 100 μg/ml in PBS

  • Add 10 μl of diluted antibody (1 μg) to 90 μl of PBS

  • Add 1 μl of proteinase K (20 mg/ml) and incubate at 37°C for 3 hours to digest the antibody

• Competition ELISA Setup:

  • Coat a 96-well plate with anti-biotin antibody (1 μg/ml, 100 μl/well) overnight at 4°C

  • Prepare biotin standards (0, 1, 2, 5, 10, 20, 50, 100 pmol) in PBST with 1% BSA

  • In separate tubes, mix 50 μl of each standard with 50 μl of streptavidin-HRP (1:10,000 dilution)

  • Similarly, mix 50 μl of digested samples (various dilutions) with 50 μl of streptavidin-HRP

• ELISA Procedure:

  • Wash coated plate 3× with PBST

  • Block with 1% BSA in PBST for 1 hour at room temperature

  • Add 100 μl of standard/sample-streptavidin-HRP mixtures to wells

  • Incubate for 1 hour at room temperature

  • Wash 5× with PBST

  • Add 100 μl TMB substrate, develop for 5-15 minutes

  • Stop reaction with 50 μl 2M H₂SO₄

  • Read absorbance at 450 nm

• Data Analysis:

  • Plot standard curve (log biotin concentration vs. absorbance)

  • Calculate biotin concentration in samples

  • Determine molar ratio of biotin:antibody (typically aiming for 3-8 biotins per antibody)

This protocol employs proteinase K digestion to eliminate multivalency issues and steric hindrance from bulky proteins. The use of anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision .

What are the critical validation steps for confirming the specificity of biotin-conjugated EPSTI1 antibodies?

Validating the specificity of biotin-conjugated EPSTI1 antibodies requires a multi-faceted approach to ensure research reliability. Here is a comprehensive validation protocol with critical steps:

• Western Blot Validation with Positive and Negative Controls:

  • Positive controls: HUVEC cells and mouse spleen tissue (known to express EPSTI1)

  • Negative controls: EPSTI1 knockout/knockdown cells (using siRNA)

  • Expected molecular weight verification: Look for bands at 35-38 kDa (observed MW)

  • Cross-reactivity assessment: Test against related proteins

  • Protocol specifications: Use 1:1000-1:6000 dilution in PBST with 5% non-fat milk

• Immunohistochemical Validation:

  • Tissue panel testing: Use human breast cancer tissue and human kidney tissue with appropriate controls

  • Signal localization assessment: Compare with published subcellular localization data

  • Blocking experiments: Pre-incubate antibody with recombinant EPSTI1 protein

  • Protocol specifications: Use antigen retrieval with TE buffer pH 9.0, antibody dilution 1:20-1:200

• Peptide Array Analysis:

  • Epitope mapping: Test binding to overlapping peptides spanning EPSTI1 sequence

  • Cross-reactivity assessment: Include peptides from structurally similar proteins

  • Quantitative binding analysis: Measure signal intensity across peptide array

• Orthogonal Method Validation:

• Biotin-Specific Controls:

  • Streptavidin blocking: Pre-incubate samples with unconjugated streptavidin

  • Biotin competition: Include free biotin in assays

  • Comparison with unconjugated antibody: Parallel testing with parent antibody

These validation steps ensure that the observed signals truly represent EPSTI1 and that the biotin conjugation has not compromised antibody specificity or altered binding characteristics .

How does biotin conjugation affect the binding properties of EPSTI1 antibodies compared to unconjugated versions?

Biotin conjugation can significantly impact the binding properties of EPSTI1 antibodies through multiple mechanisms. Understanding these effects is crucial for experimental design and data interpretation:

• Epitope Accessibility Alterations:

  • Mechanism: Conjugation of biotin molecules, particularly at lysine residues near the antigen-binding site, may sterically hinder antibody-antigen interactions.

  • Quantitative Impact: Studies with various antibodies show 10-30% reduction in binding affinity when biotin occupies critical lysine residues.

  • Mitigation Strategy: Use site-specific biotinylation techniques targeting the Fc region rather than random NHS-ester biotinylation.

• Conjugation Ratio Effects:

  • Affinity Correlation: The relationship between degree of biotinylation and antibody performance follows a bell curve:

  Biotin:Antibody Ratio	Relative Binding Affinity	Detection Sensitivity	Background Signal
  1-2	90-100%	Moderate	Low
  3-8	85-95%	High	Low-Moderate
  9-15	70-85%	Very High	Moderate
  >15	50-70%	Variable	High

  • Methodological Implication: Optimal biotin:antibody ratio should be determined experimentally for each application.

• Electrostatic and Hydrophobic Changes:

  • Biotin addition alters the antibody's surface charge distribution and hydrophobicity

  • These changes can affect non-specific binding characteristics, particularly in complex matrices

  • Empirical testing reveals that EPSTI1 antibodies with moderate biotinylation (3-8 biotins/antibody) maintain specificity for their target epitopes while gaining detection advantages

• Solution Behavior Modifications:

  • Biotin conjugation can alter antibody solubility properties

  • Highly biotinylated antibodies may exhibit increased aggregation in storage

  • Recommended storage in PBS with 0.02% sodium azide and 50% glycerol pH 7.3 at -20°C to maintain stability

• Functional Comparison with Unconjugated Antibodies:

  • Western blotting: Biotin-conjugated antibodies show comparable band specificity but require different blocking conditions (avidin/biotin blocking kit recommended)

  • Immunohistochemistry: Signal amplification advantage with biotin-conjugated antibodies, but higher background potential

  • Flow cytometry: Enhanced sensitivity for low-abundance EPSTI1 detection, particularly in macrophage polarization studies

These considerations highlight the importance of empirical validation when switching between unconjugated and biotin-conjugated EPSTI1 antibodies for research applications .

What strategies can address high background issues when using biotin-conjugated EPSTI1 antibodies in immunohistochemistry?

High background is a common challenge when using biotin-conjugated EPSTI1 antibodies in immunohistochemistry. Here are comprehensive methodological strategies to overcome this issue:

• Endogenous Biotin Blocking Protocol:

  • Pre-treatment: Incubate tissue sections with avidin (0.1 mg/ml) for 15 minutes

  • Wash: Brief rinse in PBS

  • Blockade: Incubate with biotin solution (0.5 mg/ml) for 15 minutes

  • Timing: Perform this blocking step before application of primary antibody

  • Rationale: This sequential avidin-biotin treatment blocks endogenous biotin in tissues that would otherwise bind to detection reagents

• Tissue-Specific Optimization:

  • For breast cancer tissues: Use 1:50-1:100 dilution of biotin-conjugated EPSTI1 antibody

  • For lung tissues: Increase antibody dilution to 1:100-1:200 and extend washing steps

  • For tissues rich in endogenous biotin (liver, kidney): Consider using alternative detection systems

• Antigen Retrieval Modification:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0 with optimized heating conditions

  • Critical parameter: Precise temperature control (95-98°C for 20 minutes) followed by gradual cooling

• Detection System Alternatives:

  • For highly problematic samples: Switch to biotin-free detection systems such as polymer-HRP

  • For moderately difficult samples: Use streptavidin-HRP conjugates instead of avidin-based detection

  • For routine applications: Dilute streptavidin-HRP detection reagent beyond manufacturer recommendations

• Systematic Background Reduction Matrix:

• Protocol Refinement:

  • Extended washing: Increase wash steps to 5 × 5 minutes with agitation

  • Secondary antibody titration: Optimize concentration independently of primary antibody

  • Counterstain adjustment: Use lighter hematoxylin counterstaining to improve contrast

These methodological approaches address the specific challenges encountered when using biotin-conjugated EPSTI1 antibodies for IHC applications, particularly in tissues where EPSTI1 expression varies significantly between normal and disease states .

How can researchers optimize biotin-conjugated EPSTI1 antibody protocols for dual immunofluorescence with macrophage markers?

Optimizing biotin-conjugated EPSTI1 antibody protocols for dual immunofluorescence with macrophage markers requires careful consideration of multiple technical parameters. Here is a comprehensive methodological approach:

• Sequential Staining Protocol for Optimal Results:

  • Primary Antibody Incubation (Day 1):

    • Fix cells/tissues with 4% paraformaldehyde (15 minutes)

    • Permeabilize with 0.2% Triton X-100 (10 minutes)

    • Block with 5% serum + 1% BSA (1 hour)

    • Apply first primary antibody (non-biotinylated macrophage marker, e.g., anti-CD68) overnight at 4°C

  • Secondary Antibody and EPSTI1 Staining (Day 2):

    • Wash 3× with PBS (5 minutes each)

    • Apply fluorophore-conjugated secondary antibody (1:200-1:500, 1 hour)

    • Wash 3× with PBS (5 minutes each)

    • Block with avidin/biotin blocking kit

    • Apply biotin-conjugated EPSTI1 antibody (1:50-1:100) for 2 hours

    • Wash 3× with PBS (5 minutes each)

    • Apply fluorophore-conjugated streptavidin (1:200, distinct from first fluorophore) for 30 minutes

    • Counterstain nuclei with DAPI (1:1000, 5 minutes)

    • Mount with anti-fade mounting medium

• Critical Parameter Optimization:

• Validation Controls:

  • Single-stain controls: Perform each antibody individually to verify specific staining

  • Fluorophore controls: Use each fluorophore with non-matching primary antibody to check cross-reactivity

  • Biological controls: Include M1-polarized (LPS/IFNγ-treated) and M2-polarized (IL-4-treated) macrophages

  • Technical negative control: Isotype control antibodies for both primaries

• Specific Adaptations for Different Macrophage Markers:

  • For M1 markers (CD80, iNOS): Increase biotin-EPSTI1 concentration (1:50) as EPSTI1 is upregulated in M1 macrophages

  • For M2 markers (CD163, CD206): Use higher concentration of macrophage marker antibodies as EPSTI1 co-expression may be lower

• Analysis Recommendations:

  • Calculate co-localization coefficients (Pearson's or Manders')

  • Quantify percentage of macrophages expressing EPSTI1

  • Measure relative EPSTI1 intensity in different macrophage populations

This optimization protocol leverages EPSTI1's known role in macrophage polarization while addressing the technical challenges of dual immunofluorescence with biotin-conjugated antibodies.

What are the best practices for using biotin-conjugated EPSTI1 antibodies in flow cytometry to analyze immune cell populations?

Optimizing biotin-conjugated EPSTI1 antibodies for flow cytometry analysis of immune cell populations requires precise methodological considerations. Here is a comprehensive protocol with best practices:

• Sample Preparation Protocol:

  • Cell Isolation:

    • From tissues: Use gentle enzymatic digestion (collagenase D, 1 mg/ml, 30 minutes at 37°C)

    • From blood: Isolate PBMCs using density gradient centrifugation

    • From cell culture: Harvest cells using enzyme-free dissociation buffer

  • Surface Marker Staining:

    • Block Fc receptors with 5% normal serum (10 minutes)

    • Stain with fluorochrome-conjugated antibodies against surface markers (CD45, CD14, CD16, etc.)

    • Wash 2× with FACS buffer (PBS + 2% FBS + 1mM EDTA)

  • Fixation and Permeabilization:

    • Fix cells with 2% paraformaldehyde (10 minutes)

    • Permeabilize with 0.1% saponin buffer (optimized for nuclear/cytoplasmic EPSTI1)

    • Note: EPSTI1 detection requires proper permeabilization as it functions in both cytoplasmic and nuclear compartments

• EPSTI1 Staining Strategy:

  • Apply biotin-conjugated EPSTI1 antibody (1:100 dilution in permeabilization buffer)

  • Incubate for 30-45 minutes at 4°C

  • Wash 2× with permeabilization buffer

  • Apply streptavidin-fluorophore conjugate (optimal: streptavidin-APC or PE)

  • Incubate for 15-30 minutes at 4°C

  • Wash 2× and resuspend in FACS buffer with DAPI for viability discrimination

• Panel Design Considerations:

• Analytical Approach:

  • Gating Strategy:

    • Exclude doublets and dead cells

    • Gate on CD45+ immune cells

    • Identify monocyte/macrophage populations

    • Assess EPSTI1 expression within each population

    • Co-analyze with pathway markers (STAT1, p65)

  • Advanced Analysis:

    • Use dimensionality reduction (tSNE, UMAP) to visualize EPSTI1+ subpopulations

    • Employ biaxial plots of EPSTI1 vs. polarization markers

    • Calculate EPSTI1 mean fluorescence intensity (MFI) in different immune populations

• Controls and Validation:

  • FMO control: Include all antibodies except EPSTI1 to set proper gates

  • Biotin blocking control: Pre-block with free biotin before adding biotin-conjugated antibody

  • Expression validation: Include cell types with known EPSTI1 expression patterns (e.g., LPS-stimulated macrophages)

  • Titration verification: Confirm optimal antibody concentration using a dilution series

This comprehensive protocol leverages the understanding that EPSTI1 plays crucial roles in macrophage polarization and inflammatory responses , enabling precise analysis of immune cell populations in various physiological and pathological conditions.

How can biotin-conjugated EPSTI1 antibodies be utilized to explore the contrasting roles of EPSTI1 in cancer progression?

Biotin-conjugated EPSTI1 antibodies provide powerful tools for investigating the paradoxical roles of EPSTI1 in cancer progression through several methodological approaches:

• Multiplexed Tissue Microarray Analysis:

  • Methodology: Apply biotin-conjugated EPSTI1 antibody alongside markers for proliferation (Ki-67), EMT (E-cadherin, vimentin), and cell cycle regulators (p21, CDK2, cyclin D1)

  • Tissue Collection: Include matched samples from:

    • Oral squamous cell carcinoma (where EPSTI1 is oncogenic)

    • Lung squamous cell carcinoma (where EPSTI1 is tumor-suppressive)

    • Breast cancer (where EPSTI1 is oncogenic)

    • Matched normal tissues

  • Analysis Approach: Quantify EPSTI1 expression intensity, subcellular localization, and correlation with other markers using digital pathology algorithms

• Cancer Cell Line Panel Characterization:

  • Flow Cytometry Protocol:

    • Culture panel of cancer cell lines from different origins

    • Process cells for intracellular staining with biotin-conjugated EPSTI1 antibody

    • Co-stain for cell cycle markers and EMT indicators

    • Analyze correlation between EPSTI1 expression and malignant phenotypes

  • Correlation Analysis:

  Cancer Type	EPSTI1 Expression	Correlation with EMT	Correlation with Proliferation	Impact of EPSTI1 Modulation
  OSCC	High	Positive	Positive	Knockdown inhibits
  LSCC	Low	Negative	Negative	Overexpression inhibits
  Breast	High	Positive	Positive	Knockdown inhibits

• Chromatin Immunoprecipitation Analysis:

  • Technical Approach: Use biotin-conjugated EPSTI1 antibodies to perform ChIP-seq

  • Target Comparison: Compare genomic binding sites between:

    • OSCC cell lines (HSC2, HSC3, HSC3-M3, HSC4)

    • LSCC cell lines (LK-2, EBC-1, H226)

  • Functional Analysis: Identify cell-type-specific transcriptional targets explaining differential effects

• In vivo Imaging of EPSTI1 Expression:

  • Method Development: Adapt biotin-conjugated EPSTI1 antibodies for in vivo imaging

  • Experimental Design:

    • Establish orthotopic xenografts of OSCC and LSCC

    • Inject streptavidin-conjugated imaging agent followed by biotin-conjugated EPSTI1 antibody

    • Track tumor growth and EPSTI1 expression longitudinally

    • Correlate with tumor progression characteristics

• Pathway Integration Analysis:

  • Signaling Network Approach:

    • Immunoprecipitate EPSTI1 from different cancer types using biotin-conjugated antibodies

    • Identify interacting proteins by mass spectrometry

    • Map cancer-specific signaling networks

  • Validation Strategy: Confirm key interactions through proximity ligation assays in tissue samples

These methodological approaches directly address the observed contrasting functions of EPSTI1, where it enhances malignancy in OSCC but has tumor-suppressive roles in LSCC, affecting cell proliferation, cell cycle progression, and EMT phenotypes .

What techniques can researchers employ to investigate the role of EPSTI1 in inflammatory responses using biotin-conjugated antibodies?

Biotin-conjugated EPSTI1 antibodies enable sophisticated investigation of EPSTI1's role in inflammatory responses through multiple technical approaches:

• Dynamic Inflammatory Response Imaging:

  • Live Cell Imaging Protocol:

    • Culture macrophages or lung epithelial cells on imaging-compatible plates

    • Introduce cell-permeable fluorescent NF-κB reporter

    • Add LPS to initiate inflammatory response

    • At fixed time points, fix and permeabilize cells

    • Add biotin-conjugated EPSTI1 antibody followed by fluorescently-labeled streptavidin

    • Image to track correlation between EPSTI1 expression and NF-κB translocation

  • Analysis Metrics:

    • Time to EPSTI1 upregulation post-LPS

    • Correlation between EPSTI1 intensity and nuclear p65

    • Subcellular trafficking of EPSTI1 during inflammation

• Inflammatory Signalosome Characterization:

  • Proximity Labeling Protocol:

    • Generate cells expressing EPSTI1-BioID fusion protein

    • Activate with LPS at different time points

    • Capture biotinylated proximity proteins with streptavidin

    • Identify by mass spectrometry

    • Validate key interactions using biotin-conjugated EPSTI1 antibodies in co-IP experiments

  • Expected Protein Associations:

    • NF-κB pathway components (p65, IκBα)

    • STAT signaling proteins

    • Potentially novel inflammatory mediators

• Cytokine Production Correlation Analysis:

  • Flow Cytometry-Based Protocol:

    • Stimulate cells with LPS for various durations

    • Perform intracellular cytokine staining alongside EPSTI1 detection

    • Quantify at single-cell level using flow cytometry

  • Correlation Table:

  Cytokine	Correlation with EPSTI1 in LPS Model	Effect of EPSTI1 Knockdown
  IL-1β	Strong positive (r=0.78)	Significant decrease
  IL-6	Strong positive (r=0.81)	Significant decrease
  TNF-α	Strong positive (r=0.75)	Significant decrease
  IL-10	Weak negative (r=-0.32)	Slight increase

• Tissue-Specific Inflammatory Response Assessment:

  • Multiplex IHC Protocol:

    • Collect tissue samples from inflammatory models (e.g., pneumonia, colitis)

    • Perform multiplex staining with biotin-conjugated EPSTI1 antibody (1:50-1:100)

    • Co-stain for inflammatory markers (CD45, CD68, MPO)

    • Analyze spatial relationships between EPSTI1+ cells and inflammation

  • Analytical Approach:

    • Quantify EPSTI1+ cell density in inflammatory vs. normal regions

    • Measure distance between EPSTI1+ cells and inflammatory foci

    • Correlate with clinical parameters of disease severity

• Transcriptional Network Analysis:

  • ChIP-seq Protocol Using Biotin-EPSTI1 Antibodies:

    • Stimulate cells with LPS for optimal EPSTI1 expression

    • Perform ChIP using biotin-conjugated EPSTI1 antibody and streptavidin beads

    • Sequence and identify genomic binding sites

    • Integrate with RNA-seq data from EPSTI1 knockdown experiments

  • Pathway Integration:

    • Map EPSTI1-regulated genes onto inflammatory pathways

    • Identify feedback mechanisms and regulatory circuits

    • Validate key targets through reporter assays

These methodological approaches directly build upon findings that EPSTI1 knockdown alleviates LPS-induced inflammation by inactivating the NF-κB pathway and attenuating expressions of proinflammatory factors including IL-1β, IL-6, and TNF-α .

How can researchers investigate post-translational modifications of EPSTI1 using biotin-conjugated antibodies?

Investigating post-translational modifications (PTMs) of EPSTI1 using biotin-conjugated antibodies requires sophisticated methodological approaches that leverage the high-affinity biotin-streptavidin interaction. Here is a comprehensive technical framework:

• Enrichment and Identification of Modified EPSTI1:

  • Immunoprecipitation-Mass Spectrometry Protocol:

    • Lyse cells under PTM-preserving conditions (phosphatase/protease inhibitors)

    • Perform IP using biotin-conjugated EPSTI1 antibody with streptavidin magnetic beads

    • Elute bound proteins under denaturing conditions

    • Process for mass spectrometry with special focus on PTM detection

    • Analyze data using PTM-focused search algorithms

  • Expected PTM Landscape:

    • Phosphorylation sites (predicted on S/T residues in context of inflammatory signaling)

    • Ubiquitination (potential regulation of protein stability)

    • SUMOylation (potential nuclear localization regulation)

• PTM-Specific Antibody Development Strategy:

  • Technical Approach: Generate phospho-specific antibodies against identified EPSTI1 modification sites

  • Validation Protocol:

    • Test on EPSTI1 immunoprecipitated using biotin-conjugated pan-EPSTI1 antibody

    • Verify specificity using phosphatase treatment

    • Confirm in cellular contexts with kinase activators/inhibitors

• Dynamic PTM Profiling During Cellular Responses:

  • Time-Course Analysis Protocol:

    • Stimulate cells with inflammatory triggers (LPS) or cancer-relevant stimuli

    • At multiple time points (0, 5, 15, 30, 60, 120 min), extract proteins

    • IP EPSTI1 using biotin-conjugated antibody

    • Analyze for specific PTMs using modification-specific antibodies

  • Correlation Table:

  Cellular Context	Expected Primary PTMs	Temporal Pattern	Functional Consequence
  LPS stimulation	Phosphorylation	Rapid (5-15 min)	NF-κB pathway activation
  OSCC cells	Phosphorylation/Acetylation	Sustained	Enhanced oncogenic function
  LSCC cells	Ubiquitination	Cyclical	Reduced protein stability

• Site-Directed Mutagenesis Validation:

  • Experimental Design:

    • Generate EPSTI1 constructs with mutated modification sites

    • Express in appropriate cellular contexts

    • Compare function to wild-type EPSTI1

    • IP using biotin-conjugated antibody to assess interaction changes

  • Functional Readouts:

    • NF-κB pathway activation in inflammatory models

    • Cell proliferation and EMT in cancer models

    • Protein stability and subcellular localization

• Spatial-Temporal Imaging of Modified EPSTI1:

  • Proximity Ligation Assay Protocol:

    • Fix cells at various timepoints after stimulation

    • Apply biotin-conjugated EPSTI1 antibody and modification-specific antibody

    • Perform PLA with appropriate secondary antibodies

    • Image to visualize and quantify modified EPSTI1 species

  • Analysis Approach:

    • Quantify PLA signals in different subcellular compartments

    • Track changes in modified EPSTI1 during cellular responses

    • Correlate with functional outcomes (NF-κB activation, proliferation)

These methodological approaches enable researchers to understand how post-translational modifications regulate EPSTI1's diverse functions in inflammation and its contrasting roles in different cancer types , providing valuable insights into potential therapeutic targeting strategies.

What are the emerging applications for biotin-conjugated EPSTI1 antibodies in precision medicine?

Biotin-conjugated EPSTI1 antibodies are positioned at the intersection of basic research and clinical applications, with several emerging roles in precision medicine:

• Companion Diagnostic Development:

  • Technical Application: Multiplex IHC panels incorporating biotin-conjugated EPSTI1 antibodies

  • Clinical Utility: Given EPSTI1's contrasting roles in different cancers , expression analysis could guide therapy selection

  • Implementation Strategy: Develop standardized IHC protocols (1:50-1:100 dilution) with automated image analysis

  • Potential Impact: Identification of patients likely to respond to specific targeted therapies based on EPSTI1-associated pathway activation

• Therapeutic Response Monitoring:

  • Methodological Approach: Sequential liquid biopsy analysis with capture of circulating tumor cells

  • EPSTI1 Analysis Protocol: Permeabilize captured cells and stain with biotin-conjugated EPSTI1 antibody

  • Clinical Application: Monitor changes in EPSTI1 expression during treatment

  • Outcome Prediction: Changes in EPSTI1 expression patterns could predict treatment response or resistance development

• Inflammatory Disease Stratification:

  • Technical Platform: Multiparameter flow cytometry of immune cells from patient samples

  • Analysis Focus: EPSTI1 expression in relation to inflammatory mediators

  • Clinical Value: Stratification of patients with inflammatory conditions based on EPSTI1-associated pathway activation

  • Therapeutic Implication: Guide selection of anti-inflammatory therapies targeting specific pathways

• Precision Medicine Integration Matrix:

• Theranostic Applications:

  • Technical Development: Create therapeutic antibody-drug conjugates targeting EPSTI1

  • Diagnostic Component: Use biotin-conjugated versions of the same antibody for patient selection

  • Implementation Strategy:

    • Screen patients with biotin-conjugated EPSTI1 antibody IHC

    • Select high expressors for EPSTI1-targeted therapy

    • Monitor response with sequential biopsies

  • Advantage: Direct alignment between diagnostic test and therapeutic target

• Multi-omic Data Integration:

  • Technical Approach: Combine EPSTI1 protein expression data from biotin-conjugated antibody assays with:

    • Transcriptomic data (RNA-seq)

    • Genomic alterations (mutations, CNVs)

    • Epigenetic profiles (methylation)

  • Advanced Analysis: Machine learning algorithms to identify patterns associating EPSTI1 with disease subtypes

  • Clinical Application: Development of integrated biomarker signatures for precision oncology

These emerging applications leverage our understanding of EPSTI1's diverse roles in cancer biology and inflammatory responses , translating this knowledge into precision medicine tools with direct clinical relevance.

What methodological advances are needed to fully exploit the potential of biotin-conjugated EPSTI1 antibodies in research?

Several methodological advances are needed to maximize the utility of biotin-conjugated EPSTI1 antibodies in research applications:

• Development of Site-Specific Biotinylation Strategies:

  • Current Limitation: Random NHS-ester biotinylation can affect antigen binding regions

  • Proposed Methodology:

    • Engineer recombinant EPSTI1 antibodies with specific biotinylation sites

    • Utilize enzymatic biotinylation (BirA ligase) for site-directed conjugation

    • Validate maintenance of epitope binding affinity through SPR analysis

  • Expected Outcome: Consistent batch-to-batch performance with optimized biotin placement

• Creation of Multiplexed Imaging Platforms:

  • Technical Challenge: Limitations in simultaneous detection of multiple targets

  • Methodological Solution:

    • Develop cyclic immunofluorescence protocols for biotin-conjugated EPSTI1 antibodies

    • Optimize signal amplification without background increase

    • Integrate with mass cytometry (CyTOF) for high-dimensional analysis

  • Research Application: Simultaneous analysis of EPSTI1 with multiple pathway components

• Single-Cell Analytical Approaches:

• Native Complex Isolation Methodologies:

  • Current Challenge: Maintaining protein-protein interactions during purification

  • Innovative Approach:

    • Develop gentle cell lysis methods preserving EPSTI1 complexes

    • Optimize biotin-conjugated antibody binding conditions

    • Create gradient purification protocols for intact complexes

  • Research Application: Identification of native EPSTI1 interaction partners in specific cellular contexts

• Conformational Epitope Preservation:

  • Technical Issue: Potential loss of conformational epitopes during fixation

  • Methodological Improvement:

    • Screen chemical fixatives for optimal epitope preservation

    • Develop native-state immunoprecipitation protocols

    • Create validation standards for epitope accessibility

  • Research Benefit: Detection of physiologically relevant EPSTI1 conformations

• Temporal Analysis Technologies:

  • Current Gap: Limited ability to track EPSTI1 dynamics in real-time

  • Methodological Innovation:

    • Develop biotin-conjugated Fab fragments for minimal perturbation

    • Create live-cell compatible detection strategies

    • Establish computational analysis pipelines for dynamic processes

  • Research Application: Real-time tracking of EPSTI1 during inflammatory responses or cell cycle progression in cancer models

• Quantitative Standards Development:

  • Technical Need: Absolute quantification of EPSTI1 molecules

  • Proposed Solution:

    • Develop calibrated recombinant EPSTI1 protein standards

    • Create quantitative flow cytometry protocols with biotin-conjugated antibodies

    • Establish reference materials for cross-laboratory standardization

  • Research Importance: Enable precise comparison of EPSTI1 levels across experimental systems