PTGR2 Antibody, Biotin conjugated

What is PTGR2 and what are the applications of biotin-conjugated PTGR2 antibodies?

PTGR2 (Prostaglandin Reductase 2) is an enzyme that catalyzes the conversion of 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2 . Biotin-conjugated PTGR2 antibodies are immunological tools designed to detect this enzyme in various experimental contexts with high sensitivity due to the biotin-streptavidin interaction system.

The primary applications for biotin-conjugated PTGR2 antibodies include:

• Western Blotting (WB): For detecting PTGR2 protein expression in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of PTGR2

• Immunohistochemistry (IHC): For visualization of PTGR2 in tissue sections

• Immunofluorescence (IF): For cellular localization studies of PTGR2

When designing experiments using biotin-conjugated PTGR2 antibodies, researchers should consider the specific epitope recognition (e.g., AA 206-282) and ensure appropriate secondary detection systems compatible with biotin conjugation.

How should I design experiments using biotin-conjugated PTGR2 antibodies?

When designing experiments with biotin-conjugated PTGR2 antibodies, consider the following methodological approaches:

Sample preparation:

• For tissue samples: Use formalin-fixed, paraffin-embedded specimens with appropriate antigen retrieval methods as demonstrated in pancreatic ductal adenocarcinoma studies

• For cell lysates: Prepare whole-cell lysates with RIPA buffer containing protease and phosphatase inhibitor cocktails

Detection systems:

• For immunohistochemistry: Utilize the avidin-biotin complex immunoperoxidase method for optimal signal development

• For western blotting: Transfer proteins to PVDF membranes and block non-specific antibody binding sites with 5% skim milk in PBS containing 0.1% Tween 20 (PBST)

Controls to include:

• Positive control: Known PTGR2-expressing tissues (e.g., pancreatic cancer specimens)

• Negative control: Normal pancreatic tissues (typically PTGR2-negative)

• Technical control: Non-immune IgG to assess non-specific binding

Working dilution:
Determine the optimal working dilution through titration experiments as the ideal concentration may vary based on the specific application and experimental conditions .

How can PTGR2 antibodies be used in cancer research studies?

PTGR2 has demonstrated oncogenic properties in multiple cancer types, particularly in pancreatic cancer, making PTGR2 antibodies valuable tools for investigating cancer biology:

Detection of PTGR2 overexpression in tumors:
Immunohistochemical studies using PTGR2 antibodies have revealed that 85.5% of pancreatic ductal adenocarcinoma tissues stain positive for PTGR2 expression while adjacent normal tissues show minimal or no expression . This differential expression pattern suggests PTGR2 could serve as a potential biomarker or therapeutic target.

Investigating PTGR2-mediated mechanisms in cancer progression:
When studying PTGR2 silencing effects on pancreatic cancer cells, researchers can use PTGR2 antibodies to confirm knockdown efficiency through western blotting. Research has shown that silencing PTGR2:

• Enhances reactive oxygen species (ROS) production

• Suppresses pancreatic cancer cell proliferation

• Promotes cancer cell death through increased 15-keto-PGE2 levels

Exploration of downstream molecular pathways:
PTGR2 antibodies enable researchers to examine the relationship between PTGR2 and other proteins in cancer signaling cascades. For instance, PTGR2 silencing has been shown to suppress the expression of:

• Solute carrier family 7 member 11 (xCT)

• Cystathionine gamma-lyase (CTH)
  These proteins are important providers of intracellular cysteine for glutathione (GSH) generation, a critical antioxidative defense mechanism .

What is the role of PTGR2 in inflammatory responses and how can antibodies help study this function?

PTGR2 plays a significant role in modulating inflammatory responses, particularly through its regulation of the 15-keto-PGE2 (15k-PGE2) metabolism. Biotin-conjugated PTGR2 antibodies can help elucidate these mechanisms through various experimental approaches:

In sepsis and inflammation models:
Studies have shown that disruption of the Ptgr2 gene in mice improves survival rates under both LPS- and cecum ligation/puncture (CLP)-induced experimental sepsis . Using PTGR2 antibodies, researchers can:

• Monitor PTGR2 expression levels in various inflammatory conditions

• Correlate PTGR2 protein levels with disease severity or inflammatory markers

• Validate PTGR2 knockdown efficiency in experimental models

Studying the PTGR2/15k-PGE2/NRF2 axis:
PTGR2 knockdown results in accumulation of intracellular 15k-PGE2 in activated macrophages, which leads to:

• Reduced pro-inflammatory cytokine production in LPS-stimulated cells

• Increased levels of anti-oxidative transcription factor Nuclear factor (erythroid-2) related factor-2 (NRF2)

• Augmented anti-oxidant response element (ARE)-mediated activity

• Upregulated expression of corresponding anti-oxidant genes

Researchers can use PTGR2 antibodies in combination with other molecular tools to investigate this signaling pathway and its implications for inflammatory disease treatments.

What are the optimal conditions for using biotin-conjugated PTGR2 antibodies in various applications?

Different applications require specific optimization strategies when using biotin-conjugated PTGR2 antibodies:

For Western Blotting:

• Sample preparation: Use RIPA buffer containing protease and phosphatase inhibitor cocktails for protein extraction

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Blocking: 5% skim milk in PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temperature

• Primary antibody incubation: Dilute according to manufacturer's recommendation (typically 1:500-1:2000), incubate overnight at 4°C

• Detection: Use streptavidin-HRP or avidin-based detection systems optimized for biotin conjugates

• Controls: Include positive control (PTGR2-expressing cell line) and loading control (GAPDH, HSP70)

For Immunohistochemistry:

• Tissue preparation: Formalin-fixed, paraffin-embedded sections (5-7 μm thickness)

• Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), heat-induced

• Blocking: 3-5% normal serum (species-dependent on secondary antibody)

• Primary antibody incubation: Optimal dilution determined empirically, typically 1:100-1:500

• Detection: Avidin-biotin complex immunoperoxidase method

• Visualization: DAB (3,3'-diaminobenzidine) substrate

• Counterstain: Hematoxylin for nuclear visualization

For ELISA:

• Coating: Capture antibody at 1-10 μg/ml in appropriate buffer

• Blocking: 1-5% BSA or casein in PBS

• Sample dilution: Prepare standard curves using recombinant PTGR2 protein

• Detection: Streptavidin-HRP system optimized for biotin conjugates

• Substrate: TMB (3,3',5,5'-tetramethylbenzidine) with appropriate stop solution

• Reading: Absorbance at 450 nm with 570 nm reference wavelength

What troubleshooting strategies should be employed when working with biotin-conjugated PTGR2 antibodies?

Common issues and their solutions when working with biotin-conjugated PTGR2 antibodies include:

High background in immunoassays:

• Problem: Non-specific binding or endogenous biotin interference

• Solutions:

  • Increase blocking time or concentration (5-10% normal serum)

  • Include avidin/biotin blocking step to minimize endogenous biotin interference

  • Optimize antibody dilution (perform titration experiments)

  • Include additional washing steps with increased stringency (0.1-0.3% Tween-20)

Weak or no signal detection:

• Problem: Insufficient antigen, degraded antibody, or suboptimal detection conditions

• Solutions:

  • Verify PTGR2 expression in your sample (use positive control tissues like pancreatic cancer specimens)

  • Check antibody storage conditions (avoid repeated freeze-thaw cycles)

  • Optimize antigen retrieval methods for tissue samples

  • Increase antibody concentration or incubation time

  • Ensure detection reagents are functional (test with control antibodies)

Cross-reactivity issues:

• Problem: Non-specific binding to other proteins

• Solutions:

  • Validate antibody specificity using PTGR2-knockout or knockdown controls

  • Increase washing stringency

  • Pre-absorb antibody with recombinant proteins or peptides that might cross-react

  • Consider alternative PTGR2 antibodies targeting different epitopes

Storage and stability considerations:

• Store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Contains ProClin as a preservative (handle with appropriate precautions as it is considered hazardous)

How can I measure PTGR2 enzymatic activity and its effects on 15-keto-PGE2 metabolism?

PTGR2 enzymatic activity can be assessed through several experimental approaches:

Measuring 13,14-dihydro-15-keto-PGE2 production:

• Seed cells in appropriate culture plates

• Collect culture medium after treatment (e.g., PTGR2 silencing)

• Measure 13,14-dihydro-15-keto-PGE2 concentration using Prostaglandin E Metabolite EIA Kit

• Analyze absorbance at 405 nm

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

• Express results as relative values compared to control samples

Assessing PTGR2 knockdown effects:
When studying the functional consequences of PTGR2 modulation, researchers typically examine:

• ROS production changes:

  • Measure using fluorescent probes (e.g., DCFDA)

  • Compare between PTGR2-silenced and control cells

• Cell proliferation and death:

  • Assess using standard proliferation assays (MTT, BrdU)

  • Evaluate apoptosis markers (Annexin V, cleaved caspase-3)

• 15-keto-PGE2 accumulation:

  • Quantify using mass spectrometry or specific EIA kits

  • Correlate with downstream effects

• Antioxidant system changes:

  • Measure glutathione (GSH) levels

  • Assess expression of xCT and CTH proteins

  • Evaluate NRF2 nuclear translocation and ARE-reporter activity

How should I interpret PTGR2 expression data in cancer research and inflammatory studies?

Interpreting PTGR2 expression data requires careful consideration of several factors:

In cancer research:

• High PTGR2 expression in tumor tissues compared to adjacent normal tissues may indicate oncogenic potential. For example, 85.5% of pancreatic ductal adenocarcinoma tissues show positive PTGR2 staining while normal pancreatic tissues typically do not express PTGR2 .

• The relationship between PTGR2 expression and clinical parameters should be evaluated. Current data suggests PTGR2 staining intensity in pancreatic cancer is not significantly associated with differentiation status or clinical stage .

• PTGR2 knockdown effects on cancer cell viability, ROS production, and glutathione metabolism support its role in maintaining redox balance in cancer cells .

In inflammatory studies:

• Reduced PTGR2 expression or activity (through gene disruption or pharmacological inhibition) correlates with improved survival in experimental sepsis models .

• PTGR2 inhibition leads to 15k-PGE2 accumulation, which activates the NRF2 pathway, resulting in enhanced antioxidant responses and reduced pro-inflammatory cytokine production .

• 15k-PGE2 modifies Kelch-like ECH-associated protein 1 (Keap1) at cysteine 288 post-translationally, relieving its inhibitory effect on NRF2 .

Comparing data across experimental systems:

• Different cell lines or tissue types may exhibit varying levels of PTGR2 expression and functional significance

• Species differences should be considered when translating findings between animal models and human studies

• The dual role of PTGR2 in cancer (oncogenic) versus inflammation (pro-inflammatory) highlights the context-dependent nature of its biological functions