PTGES2 Antibody

What is PTGES2 and why is it a significant research target?

PTGES2 (Prostaglandin E Synthase 2, also known as C9orf15, GBF-1, mPGES2) is a 32 kDa membrane-associated protein belonging to the GST superfamily. It catalyzes the conversion of prostaglandin H2 (PGH2) to prostaglandin E2 (PGE2) . PTGES2 is constitutively expressed in specific cell types including striated muscle cells, neurons, hepatocytes, astrocytes, and endothelium .

The significance of PTGES2 as a research target stems from:

• Its role in inflammatory pathways and prostaglandin synthesis

• Involvement in multiple disease processes including cancer development

• Association with primary hypertrophic osteoarthropathy and colorectal cancer

• Potential role in the interferon-gamma pathway and cell redox homeostasis

Notably, there remains some debate about the precise biological function of PTGES2, with some evidence suggesting it may not function as originally thought in PGE2 synthesis but may instead catalyze degradation of PGH2 to other products .

What applications are most suitable for PTGES2 antibodies?

Based on validated research protocols, PTGES2 antibodies have demonstrated effectiveness in multiple applications:

When selecting applications, researchers should consider that PTGES2 is primarily localized to the Golgi apparatus, which can impact detection sensitivity depending on the technique used .

What considerations are important for antibody selection for different model organisms?

When selecting PTGES2 antibodies for cross-species applications, consider:

• Human: Most PTGES2 antibodies are developed against human epitopes, commonly using recombinant fusion proteins of human PTGES2 (NP_079348.1)

• Mouse: Antibodies targeting amino acids 1-377 have shown good reactivity in mouse models

• Rat: Limited options available, but some antibodies show cross-reactivity

Epitope regions to consider:

• AA 145-357 (good for human, mouse, rat cross-reactivity)

• AA 88-377 (effective for human applications)

• AA 270-377 (verified for human samples)

For optimal cross-species results, review the immunogen sequence information provided by manufacturers to ensure suitable homology with your target species .

How can PTGES2 antibodies be used to investigate prostaglandin synthesis pathways in cancer research?

PTGES2 antibodies have been crucial in elucidating the role of prostaglandin synthesis in cancer development. Research methodologies include:

• Expression profiling: Using IHC and Western blot with PTGES2 antibodies (1:1000 dilution) to compare expression levels between normal and cancer tissues

• Functional studies: Combining PTGES2 antibody-based detection with STAT3 phosphorylation analysis to investigate signaling pathway interactions. This approach has been effectively utilized in endometrial cancer research

• Subcellular localization: Tracking changes in PTGES2 localization during cancer progression using immunofluorescence with co-staining for Golgi markers

• Mechanistic investigations: Examining how PTGES2 and subsequent PGE2 production affects downstream targets like CYP17α hydroxylase in hormone-dependent cancers

For cancer studies, antibodies targeting the AA 270-377 region have shown superior specificity and low background in colorectal adenocarcinoma cell lines (SW480, COLO 205) and other cancer models .

What protocols can ensure optimal PTGES2 detection in immunohistochemistry applications?

For optimal PTGES2 detection in IHC applications, follow this validated protocol:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin (24 hours)

  • Embed in paraffin and section at 5 μm thickness

• Antigen retrieval (critical step):

  • Use heat-induced epitope retrieval with basic pH buffer (pH 9.0)

  • Heat at 95-98°C for 20 minutes

• Antibody incubation:

  • Block with 5% normal serum corresponding to secondary antibody host

  • Apply primary PTGES2 antibody at 5 μg/mL (1:100 dilution for most commercial antibodies)

  • Incubate for 1 hour at room temperature or overnight at 4°C

• Detection system:

  • HRP-polymer detection systems yield superior signal-to-noise ratio

  • DAB (3,3′-diaminobenzidine) substrate provides strong visualization of cytoplasmic PTGES2

Specific considerations:

• In kidney tissue, PTGES2 shows distinctive cytoplasmic staining in epithelial cells of convoluted tubules

• Negative controls should always include isotype-matched irrelevant antibodies

How can discrepancies in PTGES2 antibody results be reconciled when examining its biological function?

The biological function of PTGES2 has been subject to debate, with conflicting experimental results reported in the literature. To address these discrepancies:

• Multi-antibody approach:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal regions)

  • Compare monoclonal (e.g., clone 5B7) with polyclonal antibodies

• Functional validation:

  • Complement antibody-based detection with enzyme activity assays

  • Measure PGE2 production in PTGES2 knockdown/overexpression systems

• Contextual analysis:

  • Consider that PTGES2 may function differently depending on GSH levels and heme availability

  • Examine PTGES2 in concert with other pathway components (COX-1/2, PTGES1)

• Alternative function assessment:

  • Investigate the proposed alternative function of PTGES2 in catalyzing PGH2 degradation to 12(S)-hydroxy-5(Z),8(E),10(E)-heptadecatrienoic acid and malondialdehyde

When interpreting contradictory results, note that PTGES2's GSH-dependent property and precise biological role remain under active investigation .

What controls are essential when using PTGES2 antibodies in Western blot analysis?

For rigorous Western blot experiments with PTGES2 antibodies, incorporate these essential controls:

• Positive controls:

  • Validated cell lines with known PTGES2 expression (SW480, COLO 205, HepG2, A549)

  • Human heart tissue (consistently shows strong PTGES2 expression)

  • Transfected 293T cells overexpressing PTGES2 (produces strong band at predicted MW of 41.9 kDa)

• Negative controls:

  • Non-transfected 293T cells (minimal endogenous expression)

  • Isotype control antibodies at matching concentrations

  • Pre-absorption control using the immunizing peptide

• Loading controls:

  • Standard housekeeping proteins (β-actin, GAPDH)

  • Total protein staining methods (Ponceau S, SYPRO Ruby)

• Molecular weight verification:

  • Expected MW for PTGES2 is 30-32 kDa in most cell types

  • Higher MW band (~42 kDa) may represent post-translationally modified forms

Protocol specifications:

• Reducing conditions recommended for optimal detection

• PVDF membrane preferred over nitrocellulose

• Immunoblot Buffer Group 1 has shown consistent results

What sample preparation methods optimize PTGES2 detection in different subcellular compartments?

PTGES2 has been identified as an integral membrane protein primarily localized to the Golgi apparatus. To effectively detect PTGES2 in different subcellular compartments:

• Whole cell lysate preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Include 1% Triton X-100 to ensure membrane protein solubilization

• Membrane fraction enrichment:

  • Perform differential centrifugation (100,000 × g)

  • Resuspend membrane pellet in buffer containing 0.5% NP-40 or 1% digitonin

• Golgi apparatus isolation:

  • Use sucrose density gradient ultracentrifugation

  • Verify Golgi enrichment with markers like GM130 or TGN46

• Immunofluorescence protocol for subcellular localization:

  • Fix cells with 4% paraformaldehyde (10 minutes)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

  • Block with 3% BSA in PBS (30 minutes)

  • Incubate with PTGES2 antibody (1:50-1:100) overnight at 4°C

  • Co-stain with organelle markers (Golgi: GM130; ER: Calnexin)

These methods enable differential analysis of PTGES2 distribution, which is particularly relevant when investigating altered localization in disease states.

How should experiments be designed to investigate PTGES2 interaction with other prostaglandin pathway proteins?

To effectively study PTGES2 interactions with other components of the prostaglandin synthesis pathway:

• Co-immunoprecipitation approach:

  • Use antibodies targeting amino acids 144-384 or 270-377 of PTGES2, which have demonstrated effectiveness in IP applications

  • Cross-link antibodies to protein A/G beads to prevent antibody contamination in eluates

  • Include reverse IP to confirm interactions (pull down with partner protein antibody)

• Proximity ligation assay (PLA):

  • Enables in situ detection of protein-protein interactions

  • Particularly useful for examining PTGES2 interactions with COX-1/2 enzymes

  • Use 1:100 dilution of PTGES2 antibody combined with antibodies against pathway partners

• FRET/BRET analysis:

  • For live-cell interaction studies

  • Requires expression of fluorescently-tagged PTGES2 and interaction partners

  • Validates interactions detected by co-IP in a cellular context

• Functional interaction studies:

  • Measure PGE2 production in systems with varying levels of PTGES2 and pathway components

  • Use specific inhibitors to dissect the contribution of individual proteins to the pathway

These methodologies provide complementary data on PTGES2 interactions, addressing both physical association and functional cooperation between pathway components.

How should researchers interpret multiple bands observed in Western blots with PTGES2 antibodies?

Multiple bands in PTGES2 Western blots are common and require careful interpretation:

• Expected band patterns:

  • Primary band at 30-32 kDa (mature PTGES2)

  • Secondary band at ~42 kDa (predicted full-length protein)

  • Lower molecular weight bands (~25 kDa) may represent proteolytic fragments

• Isoform considerations:

  • Multiple transcript variants exist for PTGES2

  • Verify which isoform your antibody targets (check epitope information)

  • Different antibody clones may preferentially detect different isoforms

• Technical solutions for band verification:

  • Peptide competition assay to identify specific bands

  • Compare results with antibodies targeting different epitopes

  • siRNA knockdown to confirm specificity of bands

  • Enrichment of subcellular fractions to identify compartment-specific forms

• Post-translational modifications:

  • Higher molecular weight bands may represent glycosylated or other modified forms

  • Treat samples with deglycosylation enzymes or phosphatases to verify

When publishing or presenting Western blot data, clearly indicate which band represents PTGES2 and provide justification based on molecular weight, knockdown validation, or other confirmatory tests.

What factors contribute to variability in PTGES2 staining patterns across different tissue types?

Researchers should consider these factors when interpreting variable PTGES2 staining patterns:

• Tissue-specific expression levels:

  • Highest expression typically observed in heart, kidney, and liver

  • Cell-type specific expression within tissues (e.g., epithelial cells in kidney convoluted tubules)

  • Expression may be upregulated in cancer tissues relative to normal counterparts

• Fixation and processing variables:

  • Overfixation can mask PTGES2 epitopes, particularly affecting membrane-associated detection

  • Antigen retrieval parameters critically affect staining intensity (basic pH buffer performs better than acidic)

  • Tissue processing time impacts preservation of Golgi structures where PTGES2 localizes

• Antibody-specific considerations:

  • Monoclonal antibodies may show more consistent staining patterns but might miss some isoforms

  • Polyclonal antibodies often show broader reactivity but potentially higher background

  • Clone 5B7 has shown consistent results across multiple tissues

• Biological variables affecting expression:

  • Inflammatory status of tissue (inflammation can upregulate PTGES2)

  • Disease state (particularly in cancer and inflammatory conditions)

  • Cell cycle phase (may affect Golgi apparatus morphology and PTGES2 distribution)

To standardize interpretation, always include positive control tissues with known PTGES2 expression patterns when evaluating new tissue types.

How can conflicting results between PTGES2 antibody detection and functional assays be reconciled?

When functional data on PTGES2 activity conflicts with antibody-based detection results, consider these approaches:

• Integrated analytical framework:

  • Combine protein detection (antibody-based) with mRNA quantification (RT-qPCR)

  • Correlate enzyme activity assays (PGE2 production) with protein expression levels

  • Evaluate protein localization in relation to activity (proper Golgi localization may be required for function)

• Context-dependent activity assessment:

  • Test PTGES2 function under varying GSH concentrations

  • Evaluate heme availability and its impact on enzymatic activity

  • Consider alternative PTGES2 functions beyond PGE2 synthesis

• Experimental validation approaches:

  • CRISPR/Cas9 knockout of PTGES2 followed by functional rescue with wild-type or mutant constructs

  • Compare results obtained with different antibody clones targeting distinct PTGES2 domains

  • Evaluate PTGES2 in multiple cellular contexts (some cell types may lack cofactors needed for activity)

• Alternative reaction analysis:

  • Measure potential alternative products of PTGES2 activity (HHT and MDA)

  • Assess PTGES2 role in PGH2 degradation rather than PGE2 synthesis

  • Investigate potential non-enzymatic functions of PTGES2 (e.g., transcriptional regulation via GATE)

This multilayered approach acknowledges that protein presence (detected by antibodies) may not always correlate with canonical enzymatic activity, particularly for proteins like PTGES2 with debated biological functions.

How can PTGES2 antibodies be utilized in cancer biomarker research?

PTGES2 antibodies can be strategically employed in cancer biomarker studies through these methodological approaches:

• Tissue microarray analysis:

  • Use validated PTGES2 antibodies (1:100-1:200 dilution) for high-throughput IHC screening

  • Correlate expression patterns with clinical parameters and outcomes

  • Examples include endometrial cancer studies showing PTGES2/PGE2 pathway activation

• Multiplex immunofluorescence:

  • Combine PTGES2 antibodies with other cancer markers for co-expression analysis

  • Use Alexa Fluor 488-conjugated PTGES2 antibodies for multicolor imaging

  • Assess relationship between PTGES2 expression and cancer stem cell markers or proliferation indices

• Circulating tumor cell (CTC) analysis:

  • Evaluate PTGES2 expression in CTCs as a potential liquid biopsy approach

  • Combine with epithelial markers for improved detection sensitivity

• Therapy response monitoring:

  • Track changes in PTGES2 expression before and after treatment

  • Investigate whether PTGES2 levels correlate with response to anti-inflammatory therapies

  • Particularly relevant for cancers where prostaglandin pathways drive progression

These applications expand the utility of PTGES2 antibodies beyond basic research into translational oncology, potentially identifying new prognostic or predictive biomarkers.

What approaches can verify PTGES2 antibody specificity in complex experimental systems?

To rigorously validate PTGES2 antibody specificity in complex experimental systems:

• Genetic validation strategies:

  • CRISPR/Cas9 knockout of PTGES2 in relevant cell lines

  • siRNA knockdown with multiple independent sequences

  • Overexpression of tagged PTGES2 to confirm co-localization with antibody staining

• Orthogonal detection methods:

  • Mass spectrometry identification of proteins in immunoprecipitated samples

  • RNA-seq correlation with protein expression patterns

  • In situ hybridization to compare mRNA localization with antibody staining

• Cross-antibody validation:

  • Test multiple antibodies targeting different epitopes of PTGES2

  • Compare results from different host species and clonality types

  • Monoclonal antibodies (clones 5B7, OTI2C3, 2C3) versus polyclonal antibodies

• Peptide competition and pre-absorption controls:

  • Pre-incubate antibody with immunizing peptide prior to application

  • Gradually increasing peptide concentration should progressively reduce specific signal

  • Particularly important for polyclonal antibodies to verify specificity

These comprehensive validation approaches ensure that observed signals truly represent PTGES2, which is essential for publishable research and therapeutic development.

How should researchers approach PTGES2 antibody-based studies in the context of STAT3 signaling pathways?

When investigating PTGES2 in relation to STAT3 signaling pathways:

• Integrated signaling analysis:

  • Use phospho-specific STAT3 antibodies alongside PTGES2 detection

  • Examine temporal relationships between STAT3 activation and PTGES2 expression

  • Evidence suggests STAT3 phosphorylation may be linked to PGE2 production in endometrial cancer

• Sequential immunoprecipitation approach:

  • First IP with PTGES2 antibody, then probe for STAT3 pathway components

  • Alternative: IP with phospho-STAT3 antibody and probe for PTGES2

  • Use antibodies targeting AA 270-377 region of PTGES2 for optimal IP results

• Chromatin immunoprecipitation (ChIP) analysis:

  • Investigate whether STAT3 binds to PTGES2 promoter regions

  • Use validated STAT3 antibodies for ChIP followed by qPCR for PTGES2 regulatory regions

  • Combine with PTGES2 expression analysis after STAT3 modulation

• Functional pathway interrogation:

  • Apply STAT3 inhibitors and measure effects on PTGES2 expression

  • Use PGE2 synthesis inhibitors and assess impact on STAT3 phosphorylation

  • Determine whether PTGES2 knockdown affects STAT3-regulated gene expression