PTGR2 Antibody

What is PTGR2 and why is it significant in research?

PTGR2 (prostaglandin reductase 2), also known as ZADH1 (zinc binding alcohol dehydrogenase domain containing 1), is a 351 amino acid cytoplasmic protein belonging to the NADP-dependent oxidoreductase L4BD family. It functions as a 15-oxoprostaglandin 13-reductase, catalyzing the NADPH-dependent conversion of 15-keto-prostaglandin E2 to 15-keto-13,14-dihydro-prostaglandin E2 .

This enzymatic activity represents a key step in the terminal inactivation of prostaglandins and is involved in the suppression of PPARγ-mediated adipocyte differentiation . PTGR2 has gained significant research interest due to its potential roles in metabolic regulation and cancer biology. Recent studies have identified PTGR2 as having oncogenic properties in gastric and pancreatic cancers, while inhibition of PTGR2 shows promise for treating metabolic disorders like diabetes .

What types of PTGR2 antibodies are available for research?

Several types of PTGR2 antibodies are commercially available, varying in characteristics that suit different experimental needs:

The selection of an appropriate antibody should be based on the specific experimental requirements, including target species, detection method, and application type .

How does the molecular structure of PTGR2 influence antibody selection?

The structural characteristics of PTGR2 are crucial for antibody selection. PTGR2 contains a LID motif and a polyproline type II helix that undergo conformational changes upon NADPH binding . Crystal structure analysis has revealed that mutation of Tyr64 and Tyr259 significantly reduces catalytic rates while increasing substrate affinity .

When selecting antibodies, researchers should consider whether they need to detect PTGR2 in its native conformation or denatured state. For applications requiring detection of functional PTGR2, antibodies targeting epitopes away from the catalytic site may be preferable. For studies examining PTGR2 activity regulation, antibodies recognizing regions near the active site might provide valuable insights into conformational changes associated with substrate binding .

What are the optimal conditions for Western blot detection of PTGR2?

For optimal Western blot detection of PTGR2, the following protocol is recommended:

• Sample preparation: Prepare whole-cell lysates with RIPA buffer containing protease and phosphatase inhibitor cocktail tablets.

• Protein quantification: Determine protein concentration using Bio-Rad Protein assay.

• Gel electrophoresis: Resolve equal amounts of protein by SDS-PAGE and transfer to PVDF membrane.

• Blocking: Block non-specific antibody binding sites with 5% skim milk in PBS containing 0.1% Tween 20 (PBST).

• Primary antibody incubation: Dilute PTGR2 antibody to 1:500-1:1000 in blocking buffer and incubate overnight at 4°C.

• Detection: PTGR2 should be detected at approximately 38-39 kDa (observed molecular weight of 38.5 kDa) .

Note that sample-dependent optimization may be necessary, and researchers should check validation data for their specific antibody .

How can I validate PTGR2 antibody specificity for my experiments?

Validating PTGR2 antibody specificity is critical for experimental reliability. A comprehensive validation approach includes:

• Positive and negative controls: Use tissues known to express PTGR2 (kidney, liver, pancreas) as positive controls. BxPC-3 cells and mouse kidney tissue have been documented as reliable positive controls . For negative controls, consider using PTGR2 knockout tissues/cells or tissues with minimal PTGR2 expression.

• Knockdown validation: Compare antibody detection between wild-type and PTGR2 knockdown/knockout samples to confirm specificity.

• Immunoprecipitation: Perform reciprocal immunoprecipitations to validate antibody-antigen interaction, as demonstrated in studies using anti-15-keto-PGE2-cysteine-BSA antibody and anti-PPARγ antibody .

• Cross-reactivity testing: If working with multiple species, test the antibody against purified recombinant proteins or lysates from different species to confirm cross-reactivity claims .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding, which should eliminate or significantly reduce the target signal.

What are the recommended protocols for immunohistochemical detection of PTGR2?

For immunohistochemical detection of PTGR2 in tissue samples, the following protocol has been validated in research studies:

• Tissue preparation: Use formalin-fixed, paraffin-embedded specimens sectioned at 4-5 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide, followed by protein blocking with 5% normal serum.

• Primary antibody: Apply mouse monoclonal or rabbit polyclonal antibody against human PTGR2 at optimized dilution (typically 1:100-1:200) and incubate overnight at 4°C.

• Detection system: Use the avidin-biotin complex immunoperoxidase method with appropriate secondary antibodies.

• Visualization: Develop with DAB substrate and counterstain with hematoxylin.

• Controls: Include negative controls using nonimmune IgG of the same isotype and concentration .

This protocol has been successfully used to demonstrate PTGR2 expression in pancreatic ductal adenocarcinoma tissues while showing absence in adjacent normal tissues .

How should I interpret PTGR2 expression patterns in normal versus disease tissues?

When interpreting PTGR2 expression patterns, consider these research-backed insights:

When inconsistencies in expression patterns arise, consider tissue heterogeneity, antibody specificity issues, or potential post-translational modifications affecting epitope recognition.

What are common troubleshooting issues with PTGR2 antibodies and how can they be resolved?

How do I correctly interpret PTGR2 functional assays in relation to antibody-based detection methods?

Integrating functional assay data with antibody-based detection requires careful consideration of several factors:

• Enzyme activity vs. protein level: PTGR2's catalytic activity (conversion of 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2) may not directly correlate with protein expression levels detected by antibodies. When discrepancies occur, consider:

  • Post-translational modifications affecting enzyme activity

  • Presence of endogenous inhibitors

  • Antibody epitope accessibility

  • Protein conformation changes affecting activity

• Functional validation: When measuring 13,14-dihydro-15-keto-PGE2 production as a readout of PTGR2 activity, use antibody detection to confirm protein expression levels. Research has shown that silencing PTGR2 reduces 13,14-dihydro-15-keto-PGE2 levels, providing a control point for validating antibody detection with functional outcomes .

• Experimental approach: A recommended protocol for correlating enzyme activity with antibody detection involves:

  • Measure 13,14-dihydro-15-keto-PGE2 using Prostaglandin E Metabolite EIA Kit per manufacturer's instructions

  • Collect cell culture medium and measure absorbance at 405 nm

  • Calculate concentration using standard curve (logit B/B0 vs. log PGEM concentration)

  • In parallel, perform Western blot to quantify PTGR2 protein levels

  • Correlate functional activity with protein expression

• Interpretation framework: When antibody detection shows high PTGR2 expression but low enzyme activity (or vice versa), investigate potential inhibitors, activators, or structural modifications affecting the protein's function.

How can PTGR2 antibodies be utilized in cancer research and what are the methodological considerations?

PTGR2 antibodies have proven valuable in cancer research, particularly for pancreatic and gastric cancers. Advanced applications include:

• Tissue microarray analysis: PTGR2 antibodies have been used to evaluate expression in large cohorts of patient samples. In a study of 76 pancreatic ductal adenocarcinoma patients, immunohistochemical staining with mouse monoclonal antibody against human PTGR2 revealed that 85.5% of tumor tissues were positive for PTGR2 expression while adjacent normal tissues were negative .

• Prognostic marker evaluation: Research has demonstrated that tumor-part PTGR2 stain intensity negatively correlates with survival in patients with intestinal-type gastric cancer, suggesting potential prognostic value. Methodology involves:

  • Carefully matched patient cohorts

  • Standardized staining protocols

  • Blinded pathologist evaluation

  • Correlation with clinical outcomes

• Therapeutic target validation: Using antibodies to confirm PTGR2 knockdown efficiency in cancer models has revealed that silencing PTGR2 enhances ROS production, suppresses cell proliferation, and promotes cell death. These findings suggest PTGR2 as a potential therapeutic target .

• Mechanism investigation: Combined use of PTGR2 antibodies with other detection methods has revealed that PTGR2 silencing affects expression of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), impacting the antioxidative defense system in cancer cells .

When applying PTGR2 antibodies in cancer research, consider tissue-specific expression patterns and validate antibody specificity in each cancer type under investigation.

What are the advanced techniques for studying PTGR2 protein-protein interactions using antibody-based approaches?

Investigating PTGR2 protein-protein interactions requires sophisticated antibody-based techniques:

• Co-immunoprecipitation (Co-IP) with validation strategy:

  • Immunoprecipitate with anti-PTGR2 antibody followed by immunoblotting for interaction partners

  • Perform reciprocal IP with partner-specific antibodies

  • Include appropriate negative controls (IgG control, knockout/knockdown samples)

  • Consider crosslinking to capture transient interactions

  Example: Research has used reciprocal immunoprecipitations to demonstrate the interaction between 15-keto-PGE2 and mPPARγ in PTGR2-related signaling pathways .

• Proximity Ligation Assay (PLA):

  • Use primary antibodies against PTGR2 and potential interaction partners

  • Apply species-specific PLA probes

  • Amplify signal only when proteins are within 40 nm proximity

  • Quantify interaction signals in different cellular compartments

  This technique is particularly valuable for detecting PTGR2 interactions in their native cellular context.

• FRET/BRET analysis with antibody validation:

  • Express fluorophore-tagged PTGR2 and interaction partners

  • Validate protein functionality using antibody detection

  • Measure energy transfer as indication of protein proximity

  • Correlate FRET/BRET signals with antibody-based quantification

  This approach allows real-time monitoring of PTGR2 interactions in living cells.

• ChIP-seq for transcriptional complex analysis:

  • Use PTGR2 antibodies to immunoprecipitate chromatin complexes

  • Identify DNA binding sites and protein partners

  • Validate findings with sequential ChIP using antibodies against interaction partners

  This technique is particularly relevant for investigating PTGR2's role in transcriptional regulation through PPARγ pathways.

How can PTGR2 antibodies be used to investigate post-translational modifications?

Investigating post-translational modifications (PTMs) of PTGR2 requires specialized antibody-based approaches:

• Phosphorylation analysis:

  • Immunoprecipitate PTGR2 using validated antibodies

  • Probe with phospho-specific antibodies or perform phospho-proteomic analysis

  • Validate findings using phosphatase treatment controls

  • Correlate with functional assays measuring PTGR2 activity

  Although specific phosphorylation sites on PTGR2 are not well-characterized in the provided research data, this approach allows identification of regulatory phosphorylation events.

• Ubiquitination/SUMOylation detection:

  • Immunoprecipitate PTGR2 under denaturing conditions

  • Probe with anti-ubiquitin or anti-SUMO antibodies

  • Use proteasome inhibitors to stabilize modified forms

  • Perform reciprocal IP to confirm modifications

  This approach helps investigate PTGR2 stability regulation and turnover mechanisms.

• Redox modification analysis:

  • Use non-reducing versus reducing conditions in protein preparation

  • Employ antibodies recognizing oxidized protein forms

  • Correlate with functional assays under oxidative stress conditions

  Given PTGR2's role in redox-sensitive pathways and its impact on ROS-mediated cell death, this approach is particularly relevant .

• Glycosylation assessment:

  • Treat samples with glycosidases before Western blotting

  • Compare molecular weight shifts using PTGR2 antibodies

  • Confirm with lectin-based detection methods

  This method helps identify potential glycosylation that might affect PTGR2 function or localization.

How are PTGR2 antibodies being used in metabolic disease research?

Recent advances in PTGR2 research have revealed its significant role in metabolic regulation, with antibody-based detection methods providing critical insights:

• Diabetes and obesity research: Studies using PTGR2 antibodies have demonstrated that PTGR2 knockout mice exhibit increased thermogenesis and resistance to diet-induced obesity. Immunoblot analysis with anti-PTGR2 antibodies has shown that loss of PTGR2 leads to increased UCP1 protein expression in all fat depots, contributing to enhanced energy expenditure .

• PPARγ signaling pathway investigations: PTGR2 antibodies have helped establish that PTGR2 inhibition increases endogenous PPARγ ligands (15-keto-PGE2), offering a novel therapeutic strategy for diabetes without relying on synthetic PPARγ ligands . The methodological approach includes:

  • Western blot detection of PTGR2 and PPARγ pathway components

  • Correlation with 15-keto-PGE2 levels measured by EIA

  • Functional validation using metabolic phenotyping

• Browning of adipose tissue: Immunoblotting with PTGR2 antibodies has revealed that PTGR2 deficiency promotes browning of adipose tissue, with increased expression of thermogenic genes. This finding provides new insights into potential therapeutic strategies for obesity .

• Insulin sensitivity improvement mechanisms: PTGR2 antibody-based studies have shown that genetic or pharmacological inhibition of PTGR2 prevents diet-induced obesity and reduces insulin resistance, positioning PTGR2 as a promising therapeutic target for metabolic disorders .

What are the emerging applications of PTGR2 antibodies in developing novel therapeutics?

PTGR2 antibodies are playing crucial roles in therapeutic development through several approaches:

• Drug screening and validation: PTGR2 antibodies enable the evaluation of compound effects on PTGR2 protein levels and activity. Research has identified that indomethacin inhibits PTGR2 with a binding mode similar to that of 15-keto-PGE2, providing a structural basis for developing selective PTGR2 inhibitors .

• Structure-based drug design: Crystal structure studies combined with antibody validation have revealed that the LID motif of PTGR2 becomes highly disordered upon inhibitor binding, indicating plasticity of the active site. This information is critical for rational inhibitor design . Key methodological steps include:

  • Protein purification and crystallization

  • Structure determination

  • Antibody-based validation of purified protein

  • Compound screening and binding analysis

• Therapeutic efficacy evaluation: PTGR2 antibodies allow researchers to monitor target engagement and pathway modulation in response to experimental therapeutics. Studies have shown that PTGR2 inhibition leads to increased 15-keto-PGE2 levels, enhanced PPARγ activity, and improved metabolic outcomes .

• Cancer therapy development: Research using PTGR2 antibodies has established PTGR2 as a putative oncogene in gastric and pancreatic cancers. Silencing PTGR2 suppresses tumor growth and induces apoptosis through ROS-mediated signaling, suggesting potential for cancer therapeutics targeting the redox status of cancer cells .

What methodological advances are needed to improve PTGR2 antibody applications in research?

Despite significant progress in PTGR2 antibody applications, several methodological challenges remain:

• Improved isoform-specific antibodies: Current antibodies may not distinguish between PTGR2 isoforms resulting from alternative splicing . Development of isoform-specific antibodies would require:

  • Identification of unique epitopes in each isoform

  • Careful immunogen design targeting isoform-specific regions

  • Extensive validation using isoform-specific knockdown models

  • Comparative analysis across tissues with different isoform expression patterns

• Antibodies targeting specific conformational states: PTGR2 undergoes conformational changes upon NADPH binding, affecting the LID motif and polyproline type II helix . Antibodies recognizing specific conformational states would enable:

  • Monitoring of activation status in different cellular contexts

  • Investigation of regulatory mechanisms controlling enzyme activation

  • Screening for conformation-selective inhibitors

• Advanced multiplexing capabilities: Developing antibody panels for simultaneous detection of PTGR2 and interacting partners would facilitate more comprehensive pathway analysis. Methodological requirements include:

  • Careful selection of compatible antibody pairs

  • Optimization of multiplex staining protocols

  • Validation in tissues with variable expression levels

  • Implementation of advanced imaging and analysis techniques

• Standardized quantification methods: Establishing reliable quantification standards for PTGR2 protein levels would improve cross-study comparisons. Approaches should include:

  • Development of recombinant protein standards

  • Validated antibody-based quantification assays

  • Inter-laboratory standardization protocols

  • Correlation with functional enzyme activity measurements