PTGR2 Antibody, FITC conjugated

What is PTGR2 and what is its primary function in cellular systems?

PTGR2 functions as a 15-oxo-prostaglandin 13-reductase, acting on multiple substrates including 15-keto-PGE1, 15-keto-PGE2, 15-keto-PGE1-alpha, and 15-keto-PGE2-alpha, with highest activity toward 15-keto-PGE2 . This enzymatic activity positions PTGR2 as a key regulator in prostaglandin metabolism pathways. Additionally, PTGR2 overexpression has been shown to repress the transcriptional activity of PPARG (Peroxisome Proliferator-Activated Receptor Gamma) and inhibit adipocyte differentiation, suggesting broader regulatory functions beyond prostaglandin metabolism .

PTGR2 is also known by several alternative names including ZADH1, PRG-2, 15-oxoprostaglandin 13-reductase, and Zinc-binding alcohol dehydrogenase domain-containing protein 1 . The protein has a calculated molecular weight of approximately 38.5 kDa and is encoded by the PTGR2 gene (UniProt primary accession number Q8N8N7) .

What considerations are important when selecting a FITC-conjugated PTGR2 antibody?

When selecting a FITC-conjugated PTGR2 antibody, researchers should consider:

• Epitope specificity: Choose antibodies targeting well-characterized epitopes. Available PTGR2 antibodies include those developed against recombinant fragment proteins (amino acids 200-300) or synthetic peptides from the N-terminal region (amino acids 65-94) .

• Validation status: Prioritize antibodies that have been validated for your specific application. While many PTGR2 antibodies are validated for Western blot and IHC-P, fluorescence applications may require additional validation .

• Host species: Rabbit polyclonal antibodies are commonly available for PTGR2 , which is important to consider when designing multi-color staining protocols to avoid cross-reactivity.

• Fluorophore properties: FITC (excitation ~495nm, emission ~519nm) is susceptible to photobleaching, so consider whether this matches your microscopy setup and experimental design.

• Species reactivity: Most available PTGR2 antibodies show reactivity against human PTGR2 , so validate cross-reactivity if studying other species.

• Application compatibility: Ensure the conjugated antibody has been validated specifically for immunofluorescence applications, as conjugation can occasionally affect antibody performance.

What are the optimal applications for FITC-conjugated PTGR2 antibodies?

FITC-conjugated PTGR2 antibodies are particularly valuable for applications requiring direct fluorescence detection without secondary antibodies. The optimal applications include:

• Flow Cytometry: Direct detection of PTGR2 in cell populations allows for quantitative analysis of expression levels across different cell types or under various treatment conditions.

• Immunocytochemistry/Immunofluorescence (ICC/IF): FITC-conjugated antibodies enable direct visualization of PTGR2 subcellular localization in fixed cells, as demonstrated with similar protein detection systems .

• Multiplex Immunofluorescence: When combined with antibodies conjugated to spectrally distinct fluorophores, FITC-conjugated PTGR2 antibodies allow simultaneous detection of multiple proteins to study co-localization or pathway relationships.

• Fluorescence-Activated Cell Sorting (FACS): For isolation of PTGR2-expressing cell populations for downstream analysis or culture.

• Confocal Microscopy: High-resolution imaging of PTGR2 distribution within cellular compartments, particularly valuable for co-localization studies with prostaglandin pathway components.

While unconjugated PTGR2 antibodies have been validated for Western blotting (recommended dilution 1/500-1/1000) and IHC-P (1/10-1/50) , FITC conjugation provides advantages for applications requiring direct fluorescence detection.

How should FITC-conjugated PTGR2 antibodies be validated before experimental use?

A comprehensive validation strategy for FITC-conjugated PTGR2 antibodies should include:

• Specificity Controls:

  • Positive control: Test in cell lines with known PTGR2 expression such as HEK-293, HepG2, and HeLa .

  • Negative control: Implement siRNA knockdown of PTGR2 to confirm signal reduction.

  • Peptide competition: Pre-absorb with immunizing peptide to verify signal specificity.

  • Mutant validation: Compare staining between wild-type and PTGR2 Y100F mutant expressing cells .

• Fluorophore-Specific Validation:

  • Photobleaching assessment: Determine signal stability under continuous illumination.

  • Autofluorescence control: Include unstained samples to establish background fluorescence levels.

  • Isotype control: Use a FITC-conjugated isotype-matched irrelevant antibody to assess non-specific binding.

• Application-Specific Controls:

  • For flow cytometry: Include single-color controls for compensation setup.

  • For microscopy: Perform Z-stack imaging to confirm complete cellular distribution patterns.

  • For multiplex applications: Include FMO (Fluorescence Minus One) controls.

• Cross-Reactivity Assessment:

  • Test for potential cross-reactivity with related proteins, particularly PTGR1.

  • Validate specificity across relevant species if performing comparative studies.

• Dilution Optimization:

  • Perform titration experiments to identify optimal antibody concentration.

  • Compare signal-to-noise ratios across different concentrations.

Thorough validation ensures reliable results and prevents misinterpretation of experimental data when using FITC-conjugated PTGR2 antibodies.

What protocol modifications are necessary when working with FITC-conjugated versus unconjugated PTGR2 antibodies?

When transitioning from unconjugated to FITC-conjugated PTGR2 antibodies, several critical protocol modifications are necessary:

• Light Protection:

  • Work under reduced ambient lighting to prevent photobleaching.

  • Cover samples with aluminum foil during all incubation steps.

  • Minimize exposure time during microscopy or flow cytometry analysis.

• Buffer Considerations:

  • Avoid buffers containing primary amines (e.g., Tris) which can quench FITC fluorescence.

  • Maintain optimal pH (7.2-8.0) for maximum FITC quantum yield.

  • Use freshly prepared buffers to ensure consistent performance.

• Antibody Concentration Adjustments:

  • FITC conjugation may affect binding efficiency, often requiring higher concentrations than unconjugated antibodies.

  • Start with recommended dilutions (based on similar applications as unconjugated antibodies: 1/50-1/200 for immunofluorescence) .

  • Perform titration experiments to determine optimal concentration.

• Incubation Parameters:

  • Consider longer incubation times at 4°C rather than room temperature.

  • Increase washing steps to reduce background fluorescence.

• Fixation Considerations:

  • PFA-based fixatives are generally preferred over methanol for preserving FITC fluorescence.

  • Optimize fixation time to balance epitope preservation and fluorescence intensity.

• Mounting Considerations:

  • Use anti-fade mounting media containing anti-photobleaching agents.

  • For flow cytometry, analyze samples immediately or store protected from light at 4°C.

• Direct vs. Indirect Protocol Differences:

  • Eliminate secondary antibody incubation and washing steps.

  • Adjust blocking protocols to account for direct antibody application.

By implementing these modifications, researchers can maximize signal quality and data reliability when working with FITC-conjugated PTGR2 antibodies.

How can researchers troubleshoot weak or non-specific signals when using FITC-conjugated PTGR2 antibodies?

When encountering signal issues with FITC-conjugated PTGR2 antibodies, systematic troubleshooting approaches can help resolve both weak signals and non-specific background:

For Weak Signal:

• Antibody Concentration Optimization:

  • Increase antibody concentration incrementally (starting from recommended dilutions of 1/50-1/100) .

  • Extend incubation time (overnight at 4°C instead of 1-2 hours).

• Sample Preparation Refinement:

  • Optimize antigen retrieval methods for fixed tissues or cells.

  • Ensure adequate permeabilization for intracellular targets.

  • Test different fixation protocols to preserve PTGR2 epitopes.

• Microscopy Settings Adjustment:

  • Increase exposure time within reasonable limits.

  • Optimize gain settings without inducing autofluorescence.

  • Use appropriate filter sets optimized for FITC (Ex: 495nm, Em: 519nm).

• Signal Enhancement Strategies:

  • Consider tyramide signal amplification compatible with FITC.

  • Evaluate alternative conjugated antibodies with brighter fluorophores.

For Non-specific Signal:

• Blocking Protocol Optimization:

  • Increase blocking agent concentration (from standard 1-5% to 10%).

  • Extend blocking time (from 30-60 minutes to 2 hours).

  • Test different blocking agents (BSA, normal serum, commercial blocking solutions).

• Washing Protocol Enhancement:

  • Increase washing duration and frequency.

  • Add detergent (0.05-0.1% Tween-20) to wash buffers.

• Antibody Dilution Adjustment:

  • Prepare dilutions in blocking buffer rather than standard buffer.

  • Pre-absorb antibody with relevant tissues/cells to reduce non-specific binding.

• Autofluorescence Reduction:

  • Add quenching steps (0.1-1% sodium borohydride or Sudan Black B treatment).

  • Implement spectral unmixing if using confocal microscopy.

• Cross-Reactivity Elimination:

  • Pre-incubate with irrelevant proteins from the same species as your sample.

  • Use more stringent washing conditions (higher salt concentration).

Systematic implementation of these approaches while changing one variable at a time will help identify and resolve signal issues when using FITC-conjugated PTGR2 antibodies.

What controls are essential when performing multicolor fluorescence experiments incorporating FITC-conjugated PTGR2 antibodies?

In multicolor fluorescence experiments using FITC-conjugated PTGR2 antibodies, a comprehensive control strategy is essential for accurate data interpretation:

• Signal Specificity Controls:

  • Isotype control: FITC-conjugated antibody of same isotype but irrelevant specificity.

  • Knockdown/knockout control: Cells with reduced or eliminated PTGR2 expression.

  • Peptide competition: Pre-absorption of PTGR2 antibody with immunizing peptide.

  • Secondary-only control: For experiments combining direct and indirect detection methods.

• Fluorophore-Specific Controls:

  • Unstained control: Sample without any antibodies to establish autofluorescence levels.

  • Single-color controls: Samples stained with each fluorophore individually to establish spectral profiles.

  • FMO (Fluorescence Minus One) controls: Samples stained with all fluorophores except FITC to identify spillover.

• Bleedthrough/Compensation Controls:

  • Spectral overlap assessment: Samples single-labeled with each fluorophore analyzed in all detection channels.

  • Compensation beads: For flow cytometry applications to set compensation parameters.

  • Sequential scanning: For confocal microscopy to minimize crosstalk between channels.

• Cross-Reactivity Controls:

  • Host species compatibility check: Ensure primary antibodies are from different species or use directly conjugated antibodies.

  • Blocking controls: Samples with normal serum from relevant species to block non-specific binding.

• Biological Reference Controls:

  • Positive tissue/cell control: Sample with known PTGR2 expression (HEK-293, HepG2, or HeLa cells) .

  • Negative tissue/cell control: Sample with minimal PTGR2 expression.

  • Treatment controls: Cells with modulated PTGR2 expression or localization.

• Co-localization Specific Controls:

  • Physical separation control: Markers known to be in distinct cellular compartments.

  • Co-localization standard: Known interacting proteins as positive control.

  • Random overlay control: Artificially combined images from separate samples to establish coincidental co-localization baseline.

A systematically implemented control strategy enhances reliability and interpretability of multicolor experiments incorporating FITC-conjugated PTGR2 antibodies.

How can FITC-conjugated PTGR2 antibodies be used to study prostaglandin metabolism pathways?

FITC-conjugated PTGR2 antibodies provide powerful tools for investigating prostaglandin metabolism through direct visualization and quantitative approaches:

• Subcellular Co-localization Studies:

  • Combine FITC-conjugated PTGR2 antibodies with differently labeled markers for organelles (ER, mitochondria) to determine the precise subcellular localization of prostaglandin metabolism.

  • Implement multi-color immunofluorescence to visualize PTGR2 in relation to other enzymes in the prostaglandin pathway (cyclooxygenases, synthases).

  • Analyze dynamic changes in localization following inflammatory stimuli or drug treatments.

• Quantitative Expression Analysis:

  • Use flow cytometry with FITC-conjugated PTGR2 antibodies to quantify expression levels across different cell populations.

  • Measure changes in PTGR2 expression following treatment with prostaglandin pathway modulators.

  • Correlate expression levels with 15-keto-PGE2 metabolism using functional assays .

• Live-Cell Dynamics:

  • If cell-permeable variants are available, monitor real-time changes in PTGR2 localization during prostaglandin pathway activation.

  • Combine with fluorescent prostaglandin analogs to visualize enzyme-substrate interactions.

• Pathway Interaction Mapping:

  • Use proximity ligation assays with FITC-conjugated PTGR2 antibodies to identify interaction partners within the prostaglandin pathway.

  • Implement FRET (Förster Resonance Energy Transfer) approaches to detect direct protein-protein interactions between PTGR2 and other pathway components.

• Tissue Distribution Analysis:

  • Apply FITC-conjugated PTGR2 antibodies in tissue microarrays to map expression patterns across multiple tissues.

  • Correlate PTGR2 expression with prostaglandin levels and inflammatory markers.

• Intracellular Trafficking Studies:

  • Track PTGR2 trafficking in response to pathway activation using time-lapse confocal microscopy.

  • Investigate how inhibitors like HHS-0701 affect PTGR2 localization and function .

By implementing these approaches, researchers can develop a comprehensive spatial and temporal understanding of PTGR2's role in prostaglandin metabolism, contributing to better characterization of this important signaling pathway.

What methods can be employed to study PTGR2 interaction with PPARG using fluorescently labeled antibodies?

Investigating the interaction between PTGR2 and PPARG (Peroxisome Proliferator-Activated Receptor Gamma) is crucial given PTGR2's reported ability to repress PPARG transcriptional activity . Several advanced fluorescence-based methods can effectively characterize this interaction:

• Proximity Ligation Assay (PLA):

  • Use FITC-conjugated PTGR2 antibody in combination with PPARG antibody and PLA probes.

  • Each detected interaction appears as a distinct fluorescent spot, allowing quantification.

  • Apply during adipocyte differentiation to track temporal changes in interaction frequency.

• Förster Resonance Energy Transfer (FRET):

  • Combine FITC-conjugated PTGR2 antibody (donor) with a compatible acceptor fluorophore-conjugated PPARG antibody.

  • Measure energy transfer efficiency as indication of proximity.

  • Implement acceptor photobleaching FRET for quantitative analysis of interaction strength.

• Fluorescence Lifetime Imaging Microscopy (FLIM):

  • Measure changes in FITC fluorescence lifetime when PTGR2 interacts with PPARG.

  • Advantage of being less sensitive to concentration variations than intensity-based FRET.

  • Provides spatial maps of interaction throughout cellular compartments.

• Co-localization Analysis:

  • Perform dual immunofluorescence with FITC-conjugated PTGR2 antibody and differently labeled PPARG antibody.

  • Apply quantitative co-localization algorithms (Pearson's, Manders' coefficients).

  • Implement super-resolution microscopy for nanoscale interaction analysis.

• BiFC (Bimolecular Fluorescence Complementation):

  • For recombinant expression systems, fuse PTGR2 and PPARG to complementary fragments of a fluorescent protein.

  • Interaction brings fragments together, restoring fluorescence.

  • Follow with antibody staining to confirm identity of interacting proteins.

• Single-Molecule Tracking:

  • Use super-resolution approaches with FITC-conjugated PTGR2 antibody fragments.

  • Track dynamic interactions with PPARG in living cells at single-molecule level.

  • Correlate interaction frequency with functional outcomes in adipocyte differentiation.

• Fluorescence Cross-Correlation Spectroscopy (FCCS):

  • Simultaneously track FITC-conjugated PTGR2 antibody and differently labeled PPARG antibody.

  • Analyze correlated movement as indication of complex formation.

  • Determine binding kinetics and complex stability in living cells.

These methodologies provide complementary approaches to characterize PTGR2-PPARG interactions, offering insights into both static and dynamic aspects of this important regulatory relationship.

How can researchers investigate PTGR2 tyrosine modifications and the effects of inhibitors using FITC-conjugated antibodies?

Investigating PTGR2 tyrosine modifications, particularly at the Y100 site targeted by inhibitors like HHS-0701 , requires sophisticated approaches combining fluorescence techniques with biochemical methods:

• Inhibitor Competition Assays:

  • Pre-treat cells with SuTEx-based inhibitors like HHS-0701 at varying concentrations .

  • Follow with FITC-conjugated PTGR2 antibody staining.

  • Quantify changes in staining pattern or intensity that might indicate conformational changes or epitope masking upon inhibitor binding.

• Co-localization with Tyrosine Modification Markers:

  • Perform dual immunofluorescence with FITC-conjugated PTGR2 antibody and antibodies against tyrosine modifications (phosphorylation, sulfation).

  • Quantify co-localization before and after treatment with tyrosine-modifying agents or inhibitors.

  • Correlate modification status with subcellular localization and enzyme activity.

• FLIM-FRET for Structural Analysis:

  • Use FITC-conjugated PTGR2 antibody in combination with probes sensitive to protein conformation.

  • Measure changes in fluorescence lifetime when Y100 site is modified or occupied by inhibitors.

  • Map structural changes throughout the protein upon modification or inhibitor binding.

• Mutant Protein Comparative Analysis:

  • Express wild-type PTGR2 and Y100F mutant in parallel samples .

  • Stain with FITC-conjugated PTGR2 antibody and quantify differences in localization or interaction patterns.

  • Assess differential sensitivity to HHS-0701 and related inhibitors.

• Activity-Based Protein Profiling Integration:

  • Combine FITC-conjugated PTGR2 antibody staining with activity-based probes like HHS-475 .

  • Compare labeling patterns before and after inhibitor treatment.

  • Correlate microscopy findings with biochemical activity data.

• Quantitative Image Analysis:

  • Implement high-content imaging to quantify multiple parameters simultaneously.

  • Measure changes in PTGR2 expression, localization, and co-localization with other factors.

  • Develop dose-response curves for inhibitor effects on these parameters.

• Flow Cytometry for Population Analysis:

  • Use FITC-conjugated PTGR2 antibody in flow cytometry to measure protein levels across cell populations.

  • Analyze population heterogeneity in response to inhibitors.

  • Sort cells based on PTGR2 expression for downstream functional analysis.

These approaches provide a comprehensive framework for understanding how tyrosine modifications, particularly at Y100, affect PTGR2 structure, interactions, and function, as well as how inhibitors like HHS-0701 modulate these properties.

What strategies can be employed to study PTGR2 expression and localization changes during cellular differentiation?

Studying PTGR2 expression and localization during cellular differentiation, particularly in adipocytes where PTGR2 inhibits differentiation through PPARG repression , requires time-resolved approaches:

• Time-Course Immunofluorescence:

  • Apply FITC-conjugated PTGR2 antibodies at defined intervals throughout the differentiation process.

  • Capture high-resolution images to track both expression level changes and subcellular redistribution.

  • Quantify fluorescence intensity changes at the single-cell level to capture population heterogeneity.

• Multiplex Phenotypic Analysis:

  • Combine FITC-conjugated PTGR2 antibody with markers of differentiation status.

  • For adipocytes, include PPARG, FABP4, and lipid droplet stains.

  • Develop multiparameter fingerprints of differentiation stages with corresponding PTGR2 expression patterns.

• Live-Cell Imaging in Differentiation Models:

  • If cell-permeable antibody fragments are available, perform continuous monitoring during differentiation.

  • Correlate dynamic PTGR2 localization changes with morphological transformations.

  • Implement photobleaching techniques to assess protein mobility changes during differentiation.

• Flow Cytometry for Population Dynamics:

  • Use FITC-conjugated PTGR2 antibody in flow cytometry at multiple timepoints.

  • Quantify population shifts in expression levels during differentiation.

  • Correlate with forward/side scatter changes indicating morphological transformation.

• Laser Capture Microdissection Integration:

  • Apply FITC-conjugated PTGR2 antibody to identify specific cell populations during differentiation.

  • Capture cells with different expression patterns for downstream molecular analysis.

  • Correlate protein localization with transcriptional profiles.

• 3D Culture Systems Analysis:

  • Implement FITC-conjugated PTGR2 antibody staining in 3D differentiation models.

  • Analyze expression patterns with respect to spatial organization and niche effects.

  • Use advanced 3D imaging techniques like light sheet microscopy for whole-sample analysis.

• Correlative Light and Electron Microscopy:

  • Use FITC-conjugated PTGR2 antibody to identify regions of interest by fluorescence.

  • Follow with electron microscopy to analyze ultrastructural features at sites of high PTGR2 expression.

  • Correlate PTGR2 distribution with subcellular changes during differentiation.

• Quantitative Image Analysis Framework:

  • Develop automated image analysis pipelines to extract multiple parameters from PTGR2 staining.

  • Track nuclear/cytoplasmic ratio, granularity, intensity, and co-localization with differentiation markers.

  • Apply machine learning algorithms to identify subtle pattern changes during differentiation.

These methodologies provide a comprehensive toolkit for characterizing PTGR2 dynamics during cellular differentiation, offering insights into its regulatory functions in developmental processes.

How should researchers analyze and interpret PTGR2 localization data from fluorescence microscopy experiments?

Analyzing and interpreting PTGR2 localization data from fluorescence microscopy requires rigorous quantitative approaches combined with biological context:

What are common pitfalls in data interpretation when using FITC-conjugated PTGR2 antibodies?

Several common pitfalls can undermine the validity of data generated using FITC-conjugated PTGR2 antibodies. Awareness of these issues and implementation of appropriate controls can enhance data reliability:

Awareness of these pitfalls and implementation of appropriate controls will enhance the reliability of data generated using FITC-conjugated PTGR2 antibodies.

How can researchers reconcile contradictory results between different antibody-based detection methods for PTGR2?

When faced with contradictory results between different PTGR2 antibody-based methods (e.g., discrepancies between FITC-conjugated antibody results and other detection approaches), a systematic reconciliation strategy is essential:

• Technical Validation Assessment:

  • Compare epitope locations between different antibodies – differences in results may reflect differential accessibility or post-translational modifications.

  • Evaluate fixation and permeabilization effects on epitope availability across methods.

  • Determine whether the FITC conjugation itself might affect antibody binding characteristics.

• Method-Specific Limitations Analysis:

  • Western Blot vs. Immunofluorescence: Recognize that denaturation in WB exposes epitopes that may be inaccessible in native conformation.

  • Flow Cytometry vs. Microscopy: Consider how whole-cell averaging differs from spatial resolution in interpreting results.

  • ELISA vs. Cell-Based Assays: Account for how protein extraction and immobilization affect epitope presentation.

• Orthogonal Validation Approaches:

  • Implement non-antibody methods (e.g., CRISPR-Cas9 tagging of endogenous PTGR2).

  • Correlate with mRNA expression data from RT-PCR or RNA-seq.

  • Use functional assays measuring 15-keto-PGE2 metabolism to validate expression data .

• Biological Variable Consideration:

  • Examine whether discrepancies correlate with specific cell types (HEK-293, HepG2, HeLa) .

  • Consider whether cell cycle stage affects results differently between methods.

  • Evaluate whether specific treatments or conditions exacerbate discrepancies.

• Reconciliation Through Integration:

  • Develop a unified model acknowledging the strengths and limitations of each method.

  • Weight evidence based on validation rigor and relevance to the biological question.

  • Implement triangulation approaches requiring convergence of multiple methods.

• Standardized Comparison Protocol:

  • Develop a systematic side-by-side comparison protocol of different methods.

  • Include identical samples processed in parallel for each method.

  • Analyze results using consistent quantification approaches.

• Consensus Development Framework:

  • Establish which aspects of results are consistent across methods.

  • Clearly communicate both agreements and discrepancies in reporting.

  • Develop operational definitions that account for method-specific differences.

By implementing these reconciliation strategies, researchers can develop a more comprehensive and accurate understanding of PTGR2 biology while acknowledging the technical limitations inherent to different methodological approaches.