PTGER3 Antibody, FITC conjugated

What is PTGER3 and what biological functions does it mediate?

PTGER3 (Prostaglandin E2 Receptor EP3 subtype) functions as a receptor for prostaglandin E2 (PGE2). This receptor's activity couples to both the inhibition of adenylate cyclase mediated by G(i) proteins and the elevation of intracellular calcium. PTGER3 is required for several critical physiological processes, including normal development of fever in response to pyrinogens (IL1B, PGE2, and bacterial lipopolysaccharide), potentiation of platelet aggregation by PGE2 (thus regulating blood coagulation), and increased bicarbonate secretion in the duodenum in response to mucosal acidification (protecting against acid-induced ulceration). Alternative names include PGE receptor EP3 subtype, PGE2 receptor EP3 subtype, PGE2-R, and Prostanoid EP3 receptor .

What are the structural characteristics of commercially available FITC-conjugated PTGER3 antibodies?

FITC-conjugated PTGER3 antibodies are typically rabbit polyclonal antibodies generated against synthetic peptides corresponding to unique amino acid sequences on the EP3 receptor protein. The antibody concentration is generally 0.5-0.6 μg/μl in antibody stabilization buffer. These antibodies are directed against specific epitopes, with some targeting the N-terminal region (amino acids 1-50) of human PTGER3. The FITC (fluorescein isothiocyanate) conjugation allows for direct fluorescent detection without the need for secondary antibodies, making these reagents particularly valuable for multicolor immunofluorescence studies .

What species reactivity can be expected with PTGER3 antibodies?

The available PTGER3 antibodies, including FITC-conjugated versions, have demonstrated reactivity with human and mouse samples. While some antibodies are specifically validated for human samples, others have cross-reactivity with mouse models. This cross-species reactivity makes these antibodies valuable for translational research comparing PTGER3 expression or localization between human clinical samples and mouse models. For other species, validation experiments should be conducted before proceeding with full-scale studies .

What are the validated applications for FITC-conjugated PTGER3 antibodies?

FITC-conjugated PTGER3 antibodies have been validated for multiple experimental applications including:

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunoprecipitation (IP)

• Confocal microscopy (CM)

Each application requires specific optimization of antibody concentration, incubation conditions, and detection parameters. The fluorescent conjugation makes these antibodies particularly suitable for applications requiring direct visualization .

What are the recommended dilution ratios for different experimental applications?

Based on available product information, the following dilution ratios are recommended:

These dilutions should be adjusted based on signal intensity and background levels observed in preliminary experiments. For applications not listed, starting with a 1:500 dilution and optimizing from there is recommended .

How should samples be prepared for optimal PTGER3 detection using immunofluorescence?

For immunofluorescence detection of PTGER3:

• Cell fixation: Use 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.2% Triton X-100 for 10 minutes

• Blocking: 5% normal serum (from the same species as the secondary antibody if using non-conjugated primary) with 1% BSA for 1 hour

• Primary antibody incubation: Apply diluted FITC-conjugated PTGER3 antibody (1:250) overnight at 4°C

• Washing: Three 5-minute washes with PBS

• Counterstaining: DAPI (1:1000) for nuclear visualization

• Mounting: Use an anti-fade mounting medium to preserve fluorescence

Tissue sections require additional steps including antigen retrieval (citrate buffer pH 6.0, heated to 95-100°C for 20 minutes) before the blocking step. Avoid using detergents that might disrupt membrane proteins like PTGER3 .

How can FITC-conjugated PTGER3 antibodies be utilized to investigate drug resistance mechanisms in ovarian cancer?

PTGER3 has been identified as a significant factor in conferring cisplatin resistance in ovarian cancer cells. Researchers can employ FITC-conjugated PTGER3 antibodies to:

• Compare PTGER3 expression levels between cisplatin-sensitive and cisplatin-resistant cell lines using quantitative immunofluorescence analysis

• Monitor changes in PTGER3 localization after drug treatment

• Perform co-localization studies with drug resistance markers or components of the Ras-MAPK/Erk2-ETS1-ELK1 signaling axis

• Analyze tumor biopsies from patients with different responses to platinum-based chemotherapy

• Track changes in PTGER3 expression during acquired resistance development

Studies have shown higher PTGER3 expression in cisplatin-resistant ovarian cancer cell lines (OVCAR5, SKOV3-TR, and A2780-CP20) compared to cisplatin-sensitive counterparts, suggesting PTGER3 as a potential biomarker for drug resistance and a therapeutic target to enhance chemosensitivity .

What signaling pathway components interact with PTGER3 and how can these interactions be visualized?

PTGER3 activates the Ras-MAPK/Erk2-ETS1-ELK1 signaling axis in cancer cells. Co-immunofluorescence studies using FITC-conjugated PTGER3 antibodies alongside antibodies against pathway components can reveal:

• Co-localization of PTGER3 with K-Ras, RAF, MEK, and Erk-2

• Activation status of downstream transcription factors ELK1 and ETS1

• Spatial relationships between receptor activation and signaling cascade components

TCGA data analysis has revealed positive correlations between PTGER3 and ETS1 (coefficient = 0.43-0.46), PTGER3 and MAPK1/Erk2 (coefficient = 0.26), and MAPK1 and ETS1 (coefficient = 0.29-0.36). These correlations support the functional relationship between these proteins, which can be further explored through high-resolution imaging of their spatial distribution and co-localization in various cellular contexts .

How can researchers utilize FITC-conjugated PTGER3 antibodies in combination with other techniques to investigate tumor microenvironments?

FITC-conjugated PTGER3 antibodies can be integrated into multiplex analysis of tumor microenvironments through:

• Multi-color immunofluorescence: Combining FITC-conjugated PTGER3 antibodies with differently labeled antibodies against CD31 (angiogenesis), Ki67 (proliferation), and TUNEL (apoptosis) markers to simultaneously assess multiple parameters in the same tissue section

• Flow cytometry: Using FITC-conjugated PTGER3 antibodies in combination with other markers to characterize and sort different cell populations from tumor samples

• Laser capture microdissection: Identifying PTGER3-expressing cells for isolation and molecular analysis

• Correlation with other inflammatory markers: Combining with COX-2 and microsomal prostaglandin E synthase (mPGES) detection to analyze the entire prostaglandin signaling axis in tumors

In vivo studies have demonstrated that silencing PTGER3 in combination with cisplatin treatment significantly reduces cell proliferation (measured by Ki67), decreases microvessel density (measured by CD31), and increases apoptosis (measured by TUNEL), highlighting the multifaceted roles of PTGER3 in the tumor microenvironment .

What quantification methods are recommended for immunofluorescence experiments using FITC-conjugated PTGER3 antibodies?

For rigorous quantification of PTGER3 expression in immunofluorescence experiments:

• Image acquisition standardization:

  • Capture multiple random fields per sample (minimum 5-10)

  • Use identical exposure settings across all experimental conditions

  • Include positive and negative controls in each imaging session

• Quantification approaches:

  • Mean fluorescence intensity (MFI) measurement in defined regions of interest

  • Percentage of PTGER3-positive cells in the population

  • Subcellular distribution analysis (membrane vs. cytoplasmic localization)

  • Co-localization coefficients (Pearson's or Mander's) when performing dual staining

• Software options:

  • ImageJ/FIJI with appropriate plugins

  • CellProfiler for automated cell-by-cell analysis

  • Commercial platforms like Definiens or Halo for tissue section analysis

• Statistical analysis:

  • Compare mean values with appropriate statistical tests (t-test or ANOVA)

  • Generate box plots or violin plots to show distribution of expression levels

  • Correlate PTGER3 expression with other experimental parameters

This quantitative approach allows for objective comparison between experimental conditions and can reveal subtle changes in expression or localization that might be missed by qualitative assessment alone .

How should researchers address potential discrepancies between mRNA and protein expression data for PTGER3?

When confronting discrepancies between PTGER3 mRNA and protein levels:

• Technical considerations:

  • Verify antibody specificity using appropriate controls

  • Confirm primer specificity for RT-PCR/qPCR

  • Check for potential splice variants that might be detected differentially

• Biological explanations:

  • Post-transcriptional regulation: miRNAs might affect translation efficiency

  • Protein stability: Differences in protein half-life can cause divergence from mRNA levels

  • Feedback mechanisms: Protein abundance might regulate mRNA transcription

• Reconciliation approach:

  • Perform time-course experiments to detect temporal relationships

  • Use protein synthesis or degradation inhibitors to assess turnover rates

  • Employ ribosome profiling to assess translation efficiency

Research has shown strong correlation between PTGER3 mRNA and protein expression in ovarian cancer cell lines (R squared = 0.850), but this correlation may not hold in all experimental systems or tissue types. Understanding the specific regulatory mechanisms in your experimental system is crucial for interpreting apparently contradictory results .

What approaches can help integrate PTGER3 expression data with functional assays to comprehensively understand its role in cancer progression?

To create an integrated understanding of PTGER3 function:

• Multi-level experimental design:

  • Expression analysis: Quantify PTGER3 levels using immunofluorescence, western blotting, and qPCR

  • Functional assays: Proliferation, migration, invasion, and drug sensitivity

  • Pathway analysis: Measure activation of downstream effectors (Ras-MAPK/Erk2-ETS1-ELK1)

  • In vivo studies: Tumor growth, metastasis, and response to therapy

• Manipulation strategies:

  • siRNA knockdown to assess loss-of-function effects

  • Overexpression systems to test gain-of-function effects

  • Selective EP3 receptor antagonists to pharmacologically inhibit function

• Data integration methods:

  • Correlation matrices between expression levels and functional outcomes

  • Principal component analysis to identify patterns across multiple parameters

  • Network analysis to place PTGER3 in the context of broader signaling networks

In ovarian cancer studies, this integrated approach revealed that PTGER3 silencing reduced tumor growth, increased cisplatin sensitivity, and altered platinum drug uptake in tumors. The effects were mediated through downregulation of the Ras-MAPK/Erk2-ETS1-ELK1 pathway and multidrug resistance proteins, demonstrating how expression data combined with functional assays can illuminate complex biological mechanisms .

What are common technical challenges when using FITC-conjugated antibodies and how can they be addressed?

Researchers frequently encounter these issues when working with FITC-conjugated PTGER3 antibodies:

• Photobleaching:

  • Use antifade mounting media containing agents like DABCO or PPD

  • Minimize exposure to light during all experimental steps

  • Capture images quickly and efficiently during microscopy sessions

  • Consider using longer-wavelength fluorophores (e.g., Alexa Fluor 488) for critical experiments

• High background fluorescence:

  • Optimize blocking conditions (try different blocking agents: BSA, serum, commercial blockers)

  • Increase washing duration and frequency

  • Use lower antibody concentration

  • Include autofluorescence quenching steps (0.1% Sudan Black B in 70% ethanol)

• Weak signal:

  • Optimize antigen retrieval for tissue sections

  • Try different fixation methods

  • Increase antibody concentration

  • Extend primary antibody incubation time

  • Use signal amplification systems (tyramide signal amplification)

• Non-specific binding:

  • Pre-adsorb antibody with tissue powder

  • Include additional blocking steps with serum from the host species

  • Validate specificity with peptide competition assays

Each of these strategies should be systematically tested and optimized for your specific experimental system and research questions .

How can researchers validate the specificity of PTGER3 antibody staining patterns?

To ensure the specificity of PTGER3 antibody staining:

• Essential controls:

  • Negative controls: Omit primary antibody; use isotype control antibody

  • Positive controls: Use cells/tissues known to express PTGER3 (e.g., cisplatin-resistant ovarian cancer cell lines)

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Genetic validation: Use PTGER3 knockout or knockdown samples

• Cross-validation approaches:

  • Compare staining patterns from different antibody clones targeting different epitopes

  • Correlate with mRNA expression data from the same samples

  • Confirm subcellular localization pattern matches known biology (primarily membrane)

  • Verify expected changes in expression under stimulation or inhibition conditions

• Technical validation:

  • Test on Western blot to confirm correct molecular weight (approximately 45-55 kDa)

  • Verify expected tissue distribution pattern (e.g., enrichment in tissues with known PTGER3 function)

Complete validation builds confidence in subsequent experimental findings and helps avoid misinterpretation of non-specific signals. For PTGER3, validation should confirm membrane localization and appropriate molecular weight, with expression patterns consistent with its biological functions .

What strategies can optimize detection of PTGER3 in different subcellular compartments?

For optimal visualization of PTGER3 across different subcellular regions:

• Membrane PTGER3 detection:

  • Use gentle fixation (2-4% paraformaldehyde, 10-15 minutes)

  • Avoid harsh detergents during permeabilization (use 0.1% saponin instead of Triton X-100)

  • Include membrane markers for co-localization (e.g., Na⁺/K⁺-ATPase)

  • Apply confocal microscopy for precise membrane resolution

• Internalized/cytoplasmic PTGER3:

  • Stimulate cells with PGE2 to induce receptor internalization

  • Use longer permeabilization (0.2% Triton X-100, 15 minutes)

  • Co-stain with endosomal markers (EEA1, Rab5) to track trafficking

  • Employ Z-stack imaging to capture the full cytoplasmic volume

• Nuclear/perinuclear PTGER3:

  • Use stronger permeabilization (0.5% Triton X-100)

  • Include nuclear membrane markers (lamin B1)

  • Apply super-resolution microscopy for precise localization

  • Complement with biochemical fractionation to confirm nuclear localization

Understanding the subcellular distribution of PTGER3 can provide insights into its signaling dynamics and potential non-canonical functions, particularly in the context of cancer cells where aberrant localization might contribute to pathological signaling .

How is PTGER3 implicated in cancer prognosis and what methodological approaches can assess its clinical value?

PTGER3 expression shows significant clinical correlations with cancer outcomes:

These findings suggest that PTGER3 assessment could potentially serve as a biomarker for identifying patients who might benefit from targeted therapies or alternative treatment strategies. Methodologically rigorous approaches to PTGER3 quantification in clinical samples are essential for its potential implementation in clinical decision-making .

What experimental designs can best investigate the therapeutic potential of targeting PTGER3 in combination with conventional chemotherapy?

To explore PTGER3 as a therapeutic target:

• In vitro experimental designs:

  • Dose-response curves with cisplatin alone vs. cisplatin + PTGER3 inhibition

  • Combination index analysis to quantify synergistic, additive, or antagonistic effects

  • Mechanistic studies examining changes in drug uptake, efflux, and DNA damage repair

  • Clonogenic survival assays to assess long-term effects of combination therapy

• In vivo approaches:

  • Orthotopic xenograft models comparing:

    • Control

    • PTGER3 silencing alone

    • Chemotherapy alone

    • Combination therapy

  • Analysis endpoints should include:

    • Tumor volume

    • Survival

    • Platinum drug uptake in tumors

    • Molecular markers (Ki67, CD31, TUNEL)

• Translational considerations:

  • Patient-derived xenografts to capture tumor heterogeneity

  • Correlative studies in clinical trials

  • Development of companion diagnostics to identify patients likely to benefit

Research has demonstrated that PTGER3 silencing (using siRNA delivery systems) combined with cisplatin significantly reduced tumor growth in mouse models, increased platinum accumulation in tumors, and enhanced the effects on proliferation, angiogenesis, and apoptosis compared to monotherapy approaches .

How can researchers integrate PTGER3 antibody studies with genomic and transcriptomic data to understand regulatory networks in cancer?

For integrated multi-omics approaches involving PTGER3:

• Correlative analyses:

  • Compare protein expression (using quantitative immunofluorescence) with mRNA levels

  • Identify genetic alterations (mutations, CNVs) that influence PTGER3 expression

  • Correlate with epigenetic modifications (methylation, histone marks)

• Network analysis approaches:

  • Identify transcription factors regulating PTGER3 expression

  • Map PTGER3-dependent gene expression signatures

  • Construct protein-protein interaction networks centered on PTGER3

• Integrative methodologies:

  • Single-cell approaches combining protein and RNA measurements

  • Spatial transcriptomics with immunofluorescence

  • ChIP-seq studies to identify binding sites for ETS1 and ELK1 on target genes

• Data mining strategies:

  • TCGA and GEO database analysis for correlations with clinical outcomes

  • Pathway enrichment analysis of PTGER3-correlated genes

  • Development of PTGER3-based gene signatures for patient stratification

Research has identified positive correlations between PTGER3 and components of the MAPK pathway, including ETS1 and MAPK1, using TCGA data analysis. Additionally, ChIP assays have confirmed the interaction between transcription factors (ETS1/ELK1) and the promoter of CFTR1, a multidrug resistance protein regulated downstream of PTGER3. These findings illustrate how integrated approaches can reveal complex regulatory networks involving PTGER3 in cancer biology .

What are the optimal storage and handling conditions for maintaining FITC-conjugated PTGER3 antibody activity?

To preserve antibody functionality and fluorescence:

• Storage conditions:

  • Long-term storage: -20°C (avoid repeated freeze-thaw cycles)

  • Working aliquots: 4°C for up to 2 weeks

  • Protect from light: Store in amber tubes or wrap in aluminum foil

  • Avoid exposure to acidic or alkaline conditions that can quench fluorescence

• Handling best practices:

  • Thaw antibodies on ice

  • Centrifuge briefly before opening tubes to collect liquid

  • Use sterile technique to prevent contamination

  • Return to appropriate storage immediately after use

• Stability considerations:

  • Monitor fluorescence intensity of positive controls over time

  • Consider adding protein stabilizers (0.1% BSA) to working dilutions

  • Document lot numbers and preparation dates

  • Validate older antibody preparations against fresh aliquots

• Transportation requirements:

  • Ship on dry ice for overnight delivery

  • Maintain cold chain throughout transportation

  • Inspect for evidence of thawing upon receipt

Proper storage and handling are essential for maintaining the specificity and sensitivity of FITC-conjugated antibodies. Given the photosensitivity of FITC, light exposure should be minimized throughout all handling steps .

How can multiplexed imaging approaches be optimized when including FITC-conjugated PTGER3 antibodies?

For effective multiplexed imaging with FITC-PTGER3 antibody:

• Fluorophore selection considerations:

  • FITC emits in the green spectrum (~520 nm)

  • Choose non-overlapping fluorophores for co-staining:

    • Red: Cy3, Alexa Fluor 594

    • Far-red: Cy5, Alexa Fluor 647

    • Blue: DAPI for nuclear counterstain

• Sequential staining protocols:

  • For multiple primary antibodies from the same host species

  • Use Zenon labeling technology or direct conjugates

  • Apply tyramide signal amplification for weak signals

• Spectral unmixing approaches:

  • Use spectral imaging systems (e.g., Zeiss LSM 880 with Airyscan)

  • Prepare single-stained controls for each fluorophore

  • Apply computational algorithms to separate overlapping emissions

• Image acquisition strategy:

  • Capture FITC channel first (most susceptible to photobleaching)

  • Minimize exposure time and laser power

  • Use sequential scanning rather than simultaneous to reduce crosstalk

This approach enables simultaneous visualization of PTGER3 with other markers such as proliferation markers (Ki67), angiogenesis markers (CD31), or signaling pathway components (phospho-ERK, ETS1) to provide comprehensive spatial information about PTGER3's role in the biological system under investigation .

What considerations are important when using FITC-conjugated PTGER3 antibodies in flow cytometry?

For optimal flow cytometry applications:

• Sample preparation optimization:

  • Cell dissociation: Use gentle methods to preserve membrane proteins

  • Fixation: 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilization: 0.1% saponin if intracellular staining is needed

  • Blocking: 2-5% serum from the same species as the antibody host

• Staining protocol considerations:

  • Antibody concentration: Start with 1:250 dilution

  • Incubation conditions: 30-60 minutes at 4°C in the dark

  • Washing: At least 3 gentle washes with PBS containing 1% BSA

  • Final resuspension: In PBS with 1% BSA and 0.1% sodium azide

• Controls and validation:

  • Unstained cells for autofluorescence assessment

  • FMO (Fluorescence Minus One) controls

  • Isotype control at the same concentration

  • Positive control (cell line with high PTGER3 expression)

  • Titration experiments to determine optimal antibody concentration

• Instrument considerations:

  • Use 488 nm laser for FITC excitation

  • 530/30 bandpass filter for emission collection

  • Compensation with single-stained controls if performing multicolor analysis

  • PMT voltage optimization for appropriate signal detection

Flow cytometry offers quantitative assessment of PTGER3 expression across cell populations and can be particularly valuable for analyzing heterogeneity in expression levels, correlating with other cellular markers, and sorting cells based on PTGER3 expression for downstream applications .

How might future developments in antibody technology enhance PTGER3 detection and functional studies?

Emerging technologies that could advance PTGER3 research include:

• Advanced antibody engineering:

  • Single-domain antibodies (nanobodies) for improved penetration and reduced size

  • Site-specific conjugation methods for optimal fluorophore positioning

  • pH-sensitive fluorescent tags to monitor receptor internalization

  • Photoactivatable antibodies for super-resolution microscopy

• Novel imaging approaches:

  • Expansion microscopy for improved spatial resolution

  • Light sheet microscopy for rapid 3D imaging with reduced photobleaching

  • Live-cell imaging with genetically encoded fluorescent protein-tagged PTGER3

  • Correlative light and electron microscopy for ultrastructural localization

• Functional antibody developments:

  • Bi-specific antibodies targeting PTGER3 and components of its signaling pathway

  • Therapeutic antibodies for targeted inhibition in cancer

  • Intrabodies for manipulation of PTGER3 in specific subcellular compartments

• High-throughput applications:

  • Tissue microarray analysis for rapid screening across multiple samples

  • Automated image analysis pipelines for standardized quantification

  • Integration with spatial transcriptomics for multilayered data

These technological advances will enable more precise detection, quantification, and functional characterization of PTGER3 in both basic research and clinical applications, potentially revealing new therapeutic opportunities in cancer and other diseases .

What are the most promising directions for translating PTGER3 research findings into clinical applications?

Translational opportunities for PTGER3 research include:

• Diagnostic and prognostic applications:

  • Development of standardized PTGER3 immunohistochemistry protocols for clinical laboratories

  • Inclusion in prognostic panels for ovarian cancer and other malignancies

  • Creation of companion diagnostics for PTGER3-targeted therapies

• Therapeutic strategies:

  • Small molecule antagonists of PTGER3

  • PTGER3-targeting antibody-drug conjugates

  • siRNA-based therapies (already demonstrated efficacy in preclinical models)

  • Combination approaches with conventional chemotherapy

• Predictive biomarker development:

  • Identification of patient subgroups likely to benefit from PTGER3 inhibition

  • Monitoring PTGER3 expression during treatment to detect resistance development

  • Integration into liquid biopsy approaches for non-invasive monitoring

• Clinical trial design considerations:

  • Stratification based on PTGER3 expression levels

  • Pharmacodynamic markers to confirm target engagement

  • Combination strategies with platinum-based chemotherapy

The association between PTGER3 overexpression and cisplatin resistance in ovarian cancer makes it a particularly promising target for enhancing chemotherapy efficacy in patients with refractory disease. Preclinical evidence demonstrates that silencing PTGER3 leads to increased platinum accumulation in tumors and enhanced therapeutic effects, providing a strong rationale for clinical translation .

What key methodological challenges remain to be addressed in PTGER3 antibody-based research?

Despite significant progress, several methodological challenges persist:

• Antibody specificity issues:

  • Distinguishing between PTGER3 splice variants

  • Cross-reactivity with other prostaglandin receptor subtypes

  • Validation across diverse experimental systems and species

  • Detection of low expression levels in certain tissues

• Technical limitations:

  • Preserving membrane protein integrity during sample preparation

  • Quantifying absolute receptor numbers rather than relative expression

  • Maintaining FITC stability during long-term storage

  • Achieving reproducible results across different laboratories

• Functional analysis gaps:

  • Correlating protein levels with receptor activity

  • Distinguishing active vs. inactive receptor conformations

  • Monitoring dynamic changes in receptor localization in real-time

  • Identifying receptor-specific signaling outputs

• Translation obstacles:

  • Standardization of protocols for clinical implementation

  • Development of reference standards for quantitative assessment

  • Integration with other biomarkers for comprehensive patient profiling

  • Validation in large, diverse patient cohorts