ptges2 Antibody

What is PTGES2 and what cellular functions does it serve?

PTGES2 (Prostaglandin E Synthase 2) is a 32 kDa member of the GST superfamily of molecules involved in prostaglandin E2 (PGE2) synthesis. It functions as a constitutively expressed, integral membrane protein embedded primarily in the Golgi apparatus. PTGES2 is found in select cell types, including striated muscle cells, neurons, hepatocytes, astrocytes, and endothelium .

Human PTGES2 is a 377 amino acid type III (no signal sequence) transmembrane protein . Its activity influences numerous signaling cascades, including those involved in immune responses and vascular functions .

Which applications are most suitable for PTGES2 antibody-based detection?

Based on validated research protocols, PTGES2 antibodies are suitable for multiple applications:

When performing Western blot analysis, researchers have successfully detected PTGES2 in multiple human cell lines, including SW480 and COLO 205 (colorectal adenocarcinoma), HepG2 (hepatocellular carcinoma), A549 (lung carcinoma), and human heart tissue . For immunohistochemistry, specific staining has been localized to the cytoplasm in epithelial cells of convoluted tubules in human kidney tissue .

What are typical cross-reactivity profiles for PTGES2 antibodies?

PTGES2 antibodies demonstrate varying cross-reactivity profiles depending on the host species and epitope target. Most commercially available antibodies show reactivity against human PTGES2, while some also cross-react with mouse and rat homologs .

For optimal experimental design, consider these reactivity patterns:

• Human-specific PTGES2 antibodies: These typically recognize epitopes in regions that are less conserved across species. Many monoclonal antibodies (e.g., clone 998012) are specifically validated for human samples only .

• Multi-species reactive antibodies: Some polyclonal antibodies recognize conserved regions and demonstrate cross-reactivity with human, mouse, and rat PTGES2 . These are particularly valuable for comparative studies across species models.

Before selecting an antibody for your experiment, verify the specific reactivity claims and, when possible, conduct preliminary validation in your experimental system with appropriate positive and negative controls .

How can researchers validate PTGES2 antibody specificity for experimental applications?

Comprehensive validation of PTGES2 antibody specificity requires multiple complementary approaches:

• Western blot analysis with recombinant controls: Compare lysates from PTGES2-transfected cells against non-transfected controls. A specific antibody should detect a band at approximately 30-32 kDa in transfected lysates with minimal background in control lysates . For example, analysis of PTGES2 expression in transfected 293T cell lines has been used successfully to confirm antibody specificity .

• Epitope mapping: Select antibodies targeting different regions of the PTGES2 protein (e.g., AA 88-377, AA 106-313, AA 145-357, AA 270-377) . Consistent detection across multiple epitope-targeted antibodies provides stronger evidence for specificity.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the immunoprecipitated protein is indeed PTGES2 rather than a cross-reactive protein .

• RNA interference: Knockdown of PTGES2 expression using siRNA should result in decreased antibody signal if the antibody is specific.

• Tissue distribution analysis: Compare antibody staining patterns with known PTGES2 expression profiles. PTGES2 shows specific expression in kidney convoluted tubules, colorectal cells, and heart tissue, which can serve as positive controls .

What methodological considerations are important when using PTGES2 antibodies for immunohistochemistry?

Successful immunohistochemistry (IHC) with PTGES2 antibodies requires careful attention to sample preparation and protocol optimization:

• Fixation and embedding: PTGES2 detection has been validated in immersion-fixed, paraffin-embedded tissue sections. Overfixation can mask epitopes, while inadequate fixation may compromise tissue morphology .

• Antigen retrieval: Heat-induced epitope retrieval using basic antigen retrieval reagents is recommended. For example, one validated protocol subjects tissue to heat-induced epitope retrieval before incubation with primary antibody . For some tissues, trypsin treatment (0.1% trypsin in 0.1% CaCl₂) for 10 minutes has also proven effective to cleave protein crosslinks and expose antigens .

• Blocking procedure: Non-specific binding should be blocked using 4% bovine serum albumin (BSA) for approximately 2 hours at room temperature .

• Antibody dilution and incubation: For monoclonal antibodies, a concentration of 5-25 μg/mL with incubation for 1 hour at room temperature has shown optimal results. For polyclonal antibodies, dilutions of 1:50-1:200 are typically effective .

• Detection system: DAB (diaminobenzidine) staining with hematoxylin counterstaining provides excellent visualization of PTGES2 in tissue sections. The Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody detection system has been successfully used with PTGES2 antibodies .

• Positive control selection: Human kidney tissue, particularly the epithelial cells in convoluted tubules, serves as an excellent positive control for PTGES2 immunostaining .

How does PTGES2 expression correlate with disease progression in cancer models?

Recent research has identified PTGES2 as a potential biomarker with significant disease associations:

• Basal Cell Carcinoma (BCC): PTGES2 has been identified as a robust protective factor for BCC, with odds ratios of 0.497 (deCODE study) and 0.494 (UKB-PPP study). Colocalization analysis has provided strong evidence of PTGES2 association with BCC (posterior probability PP.H4 >0.92) .

• Non-melanoma skin cancer (NMSC): PTGES2 shows a favorable association with NMSC development, with odds ratios of 0.476 (deCODE) and 0.472 (UKB-PPP). Interestingly, while NMSC encompasses both BCC and squamous cell carcinoma (SCC), researchers did not observe an association between PTGES2 and SCC specifically .

• Genetic associations: SNP rs13283456 has been identified as a top SNP for PTGES2. This coding non-synonymous variant within PTGES2 has shown significant association with primary graft dysfunction in previous studies .

• Expression in colorectal cancer: PTGES2 has been detected in SW480 and COLO 205 human colorectal adenocarcinoma cell lines, suggesting potential roles in colorectal cancer progression .

• Endometrial cancer correlations: Some studies indicate a notable association between increased expression of PTGES2 and age (P=0.0092) and the depth of myometrial invasion (P<0.0001) .

The emerging evidence suggests PTGES2 may serve as both a biomarker and potential therapeutic target, particularly in contexts where PGE2 signaling influences cancer progression .

What are the optimal sample preparation protocols for PTGES2 detection in different applications?

Sample preparation requirements vary by application and tissue type:

For Western Blot analysis:

• Protein extraction should be performed under reducing conditions using Immunoblot Buffer Group 1 for optimal results .

• PVDF membranes have been successfully used for protein transfer when detecting PTGES2 .

• Expected molecular weight is approximately 30-32 kDa under standard reducing conditions, though some variations (up to 38 kDa) have been reported in certain systems .

For Immunohistochemistry:

• Immersion fixation followed by paraffin embedding preserves PTGES2 antigenicity in tissue sections .

• Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic is recommended prior to primary antibody incubation .

• For some tissue types, trypsin treatment (0.1% trypsin in 0.1% CaCl₂) for 10 minutes improves epitope accessibility .

For Immunofluorescence:

• Dilution ranges of 1:50-1:100 have proven effective for detecting PTGES2 in cellular preparations .

• Blocking with 4% BSA for 2 hours at room temperature helps minimize background signal .

For ELISA:

• When using PTGES2 antibodies as capture antibodies, the detection limit for recombinant GST-tagged PTGES2 is approximately 3ng/ml .

• GST tag alone should be included as a negative control to ensure specificity .

What are common challenges in PTGES2 detection and how can they be addressed?

Researchers frequently encounter several challenges when working with PTGES2 antibodies:

• Variable molecular weight detection: PTGES2 can appear at different molecular weights (30-38 kDa) depending on the experimental system and possible post-translational modifications . Solution: Include positive controls with known PTGES2 expression, such as SW480 or COLO 205 cell lysates, to establish the correct band identification .

• Cross-reactivity concerns: Some antibodies may cross-react with other GST superfamily members. Solution: Validate antibody specificity using PTGES2-transfected versus non-transfected cell lysates , and employ antibodies targeting different epitopes to confirm consistent detection patterns .

• Low expression levels in some tissues: PTGES2 expression can be minimal in certain tissues, making detection challenging. Solution: Enhance signal using polymer-based detection systems such as the VisUCyte™ HRP Polymer Antibody system instead of traditional secondary antibodies .

• Antigen masking in fixed tissues: Formalin fixation can mask PTGES2 epitopes. Solution: Optimize antigen retrieval methods, combining both heat-induced epitope retrieval and enzymatic treatment (trypsin) when necessary .

• Non-specific background staining: This is particularly problematic in immunohistochemistry applications. Solution: Extend blocking time (up to 2 hours) with 4% BSA and optimize antibody dilutions based on each tissue type being examined .

How can researchers distinguish between different PTGES isoforms?

Several approaches can help researchers differentiate between PTGES isoforms (PTGES1/mPGES-1, PTGES2/mPGES-2, and PTGES3/cPGES):

• Epitope-specific antibodies: Select antibodies targeting unique regions not conserved across PTGES isoforms. For example, antibodies targeting amino acids 270-377 of PTGES2 recognize regions specific to this isoform .

• Molecular weight discrimination: Under reducing conditions, PTGES1 is approximately 16-18 kDa, PTGES2 is 30-32 kDa, and PTGES3 is about 17 kDa. Western blot analysis can therefore distinguish between these isoforms based on molecular weight .

• Subcellular localization studies: PTGES2 is primarily localized to the Golgi apparatus, whereas PTGES1 is associated with the microsomal fraction, and PTGES3 is predominantly cytosolic. Immunofluorescence or subcellular fractionation studies can exploit these differences .

• Expression pattern analysis: The isoforms show differential expression patterns across tissues. PTGES2 is constitutively expressed in select cell types including striated muscle cells, neurons, hepatocytes, astrocytes, and endothelium .

• Functional differences: Unlike PTGES1, PTGES2 is not a glutathione-dependent enzyme. Functional assays examining glutathione dependency can help differentiate between these isoforms .

How is PTGES2 being utilized as a biomarker in clinical research?

Recent studies have identified several promising applications for PTGES2 as a biomarker:

• Basal Cell Carcinoma (BCC) prediction: PTGES2 has been identified as a protective plasma protein biomarker for BCC, with strong evidence of colocalization with BCC based on a posterior probability PP.H4 >0.92 .

• Phenome-wide significance: MR-PheWAS analysis confirmed that BCC was the most significant phenotype associated with PTGES2 among 2,408 phenotypes in the FinnGen R10 study, highlighting its specificity as a BCC biomarker .

• Genetic variants as markers: SNP rs13283456, a coding non-synonymous variant within PTGES2, shows significant disease associations and could serve as a genetic marker for disease susceptibility .

• Cancer invasion and progression: Increased PTGES2 expression has shown associations with the depth of myometrial invasion (P<0.0001) in some cancer types, suggesting its potential as a prognostic marker for invasion and metastasis .

• Therapeutic target potential: Beyond its biomarker value, PTGES2 is being explored as a potential therapeutic target for BCC and other conditions where prostaglandin signaling plays a role in disease progression .

The clinical utility of PTGES2 as a biomarker is still emerging, but current evidence suggests it may offer valuable insights into disease mechanisms and potential intervention strategies .

What emerging techniques are enhancing PTGES2 antibody applications in research?

Several advanced techniques are expanding the utility of PTGES2 antibodies in research:

• Simple Western™ technology: This automated capillary-based immunoassay has successfully detected PTGES2 in human heart tissue and SW480 cell lysates at approximately 38 kDa, offering increased sensitivity and reproducibility compared to traditional Western blotting .

• VisUCyte™ HRP Polymer Antibody detection: This polymer-based detection system enhances signal detection in immunohistochemistry applications, improving sensitivity for PTGES2 detection in tissue sections .

• Genetic colocalization analysis: Techniques combining protein quantitative trait loci (pQTL) with genome-wide association studies (GWAS) allow researchers to determine whether PTGES2 and disease phenotypes share the same causal variation locus, strengthening evidence for their association .

• Mendelian Randomization (MR) analysis: This approach helps establish causal relationships between PTGES2 levels and disease outcomes, distinguishing biomarkers that may be causally involved in disease from those that are merely correlative .

• MR-PheWAS (phenome-wide association studies): This technique allows researchers to explore associations between PTGES2 and thousands of phenotypes simultaneously, providing a comprehensive view of its potential roles across the human disease spectrum .

These emerging techniques are enhancing both the sensitivity and specificity of PTGES2 detection while also expanding our understanding of its functional roles in health and disease.

What are the implications of PTGES2 expression in inflammation and immune response research?

PTGES2's role in prostaglandin synthesis positions it as a key player in inflammation and immune responses:

• PGE2 signaling in immune regulation: PTGES2-derived PGE2 can induce dysfunction in intratumoral cDC1s (conventional dendritic cells type 1), impairing their capacity to coordinate anti-cancer CD8+ T cell responses .

• T cell differentiation effects: PGE2 promotes Th1 differentiation and Th17 cell expansion, potentially exacerbating immune-related inflammation .

• Cancer microenvironment modulation: The effects of PTGES2 and PGE2 signaling appear to be context-dependent. For example, blocking PGE2 signaling in cancer-associated fibroblasts inhibits breast cancer growth but paradoxically promotes metastasis .

• Cyclooxygenase pathway interactions: PTGES2 operates downstream of COX-1 and COX-2, which are classic targets of non-steroidal anti-inflammatory drugs (NSAIDs). Understanding PTGES2 expression may help explain differential responses to anti-inflammatory therapies .

• Constitutive vs. inducible expression: Unlike some other components of the prostaglandin synthesis pathway that are inducible during inflammation, PTGES2 is constitutively expressed, suggesting it may maintain basal PGE2 levels under normal physiological conditions .

Research into PTGES2's immunological functions continues to evolve, with potential implications for developing targeted therapies for conditions where inappropriate inflammation contributes to disease pathology.

What are reliable positive controls for validating PTGES2 antibody performance?

When validating PTGES2 antibodies, the following positive controls have been experimentally verified:

• Cell line lysates:

  • SW480 human colorectal adenocarcinoma cells

  • COLO 205 human colorectal adenocarcinoma cells

  • HepG2 human hepatocellular carcinoma cells

  • A549 human lung carcinoma cells

  • 293T cells transfected with PTGES2 expression constructs

• Tissue samples:

  • Human heart tissue - consistently shows PTGES2 expression

  • Human kidney tissue - specifically in epithelial cells of convoluted tubules

• Recombinant proteins:

  • E. coli-derived recombinant Human PTGES2 (Glu88-His377)

  • GST-tagged PTGES2 recombinant fusion proteins

For each application, the appropriate positive control should be selected based on the expected expression level and the specific detection method being used.

What are the most effective storage and handling protocols for maintaining PTGES2 antibody activity?

To preserve PTGES2 antibody functionality and prevent degradation:

• Storage temperature:

  • Lyophilized antibodies should be stored at -20°C

  • Reconstituted antibodies in 50% glycerol can be stored at -20°C

  • For short-term storage (1-2 weeks), 4°C is acceptable for reconstituted antibodies

• Reconstitution guidelines:

  • Lyophilized antibodies should be reconstituted at 0.5 mg/mL in sterile PBS

  • Some formulations include trehalose as a stabilizing agent during lyophilization

• Aliquoting recommendations:

  • Once reconstituted, antibodies should be aliquoted to avoid repeated freeze-thaw cycles

  • Small working aliquots minimize the need to repeatedly thaw the entire stock

• Buffer composition considerations:

  • Some PTGES2 antibodies are supplied in 50% glycerol buffers for stability

  • PBS with preservatives is commonly used for diluted working solutions

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity

  • Follow manufacturer-specific recommendations, as formulations may vary

Proper storage and handling significantly extend antibody shelf-life and ensure consistent experimental results.