PAFAH2 Antibody

What is PAFAH2 and what specific biological functions does it perform?

PAFAH2 (Platelet-Activating Factor Acetylhydrolase 2) is a 40kDa enzyme that catalyzes the hydrolysis of the acetyl group at the sn-2 position of platelet-activating factor (PAF) and its analogs, leading to their inactivation . Beyond its primary PAF-hydrolyzing activity, PAFAH2 demonstrates versatile enzymatic capabilities, including hydrolyzing propionyl and butyroyl moieties at approximately half the efficiency of PAF . The enzyme also catalyzes transacetylation reactions, transferring acetyl groups from PAF to lysoplasmalogen and sphingosine, which produces plasmalogen analogs of PAF and N-acetylsphingosine (C2-ceramide) respectively . PAFAH2 exhibits marked selectivity for phospholipids containing short acyl chains at the sn-2 position, indicating a substrate specificity that differentiates it from related enzymes . Recent research has also revealed PAFAH2's crucial role in the production of omega-3 fatty acid epoxides, particularly 17,18-EpETE and 19,20-EpDPE, with significant implications for pulmonary vascular health .

How should researchers select the appropriate PAFAH2 antibody for specific experimental applications?

Selection of the appropriate PAFAH2 antibody should be guided by several methodological considerations:

• Target epitope region: Consider which region of PAFAH2 is relevant to your research question. Various antibodies target different amino acid sequences (e.g., AA 1-392, AA 1-206, AA 249-276, AA 293-392) . For studying full-length protein, antibodies targeting AA 1-392 may be optimal, whereas those targeting specific domains might be preferable for focused functional studies.

• Host species compatibility: Evaluate potential cross-reactivity issues based on your experimental system. While most available PAFAH2 antibodies show human reactivity, only some demonstrate cross-reactivity with mouse and rat samples . This is particularly important for comparative studies across species.

• Application requirements: Match the antibody to your intended application. For example:

  • For Western Blotting: Polyclonal rabbit antibodies targeting AA 1-392 show good reactivity

  • For Immunohistochemistry: Several polyclonal rabbit antibodies are validated for IHC-P applications

  • For co-localization studies: Consider antibodies validated for immunofluorescence (IF)

• Clonality considerations: Most commercial PAFAH2 antibodies are polyclonal, which offers advantages for detection sensitivity but may have batch-to-batch variation . If epitope-specific detection is critical, consider antibodies raised against shorter, specific regions.

What are the validated detection methods for PAFAH2 and their optimization parameters?

Several detection methods have been validated for PAFAH2 research, each requiring specific optimization:

• Western Blotting (WB):

  • Recommended gel concentration: 10% SDS-PAGE has been validated for PAFAH2 detection

  • Blocking conditions: 5% non-fat milk in TBST (Tris-buffered saline with Tween-20) for 1 hour at room temperature

  • Primary antibody dilution: Typically 1:500-1:2000, with overnight incubation at 4°C

  • Expected band size: Approximately 40kDa

• Immunohistochemistry (IHC):

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

  • Primary antibody dilution: 1:100-1:500, with overnight incubation at 4°C

  • Detection systems: Both DAB (3,3'-diaminobenzidine) and fluorescent secondary antibody systems are compatible

• Luminex Assay for quantitative measurement:

  • Sample types: Validated for serum, plasma, tissue homogenates, cell lysates, and cell culture supernatants

  • Equipment requirements: Luminex MAGPIX®, Luminex 100™, Luminex 200™, or Bio-Rad®, Bio-Plex® analyzer

  • Sample preparation: Requires specific diluents and buffer systems as provided in commercial kits

How does PAFAH2 contribute to omega-3 fatty acid metabolism and what are the implications for pulmonary hypertension?

PAFAH2 plays a crucial role in omega-3 fatty acid metabolism with significant implications for pulmonary vascular homeostasis:

• Epoxide production: PAFAH2 is instrumental in the production of omega-3 fatty acid epoxides, particularly 17,18-EpETE and 19,20-EpDPE, which have been identified as critical mediators in pulmonary vascular regulation .

• Knockout models: Studies in Pafah2 knockout (KO) mice have demonstrated significantly reduced production of omega-3 epoxides in the lungs under hypoxic conditions compared to wild-type mice . This reduction correlates with advanced pulmonary vascular pathology, suggesting a protective role for PAFAH2-derived omega-3 epoxides.

• Vascular remodeling: Histological analyses reveal that Pafah2 KO mice with pulmonary hypertension (PH) develop more severe pulmonary vascular remodeling, characterized by thickened pulmonary artery walls and extensive perivascular fibrosis .

• Cardiac effects: Pafah2 KO mice exhibit elevated right ventricular systolic pressure (RVSP) during cardiac catheterization, indicating increased severity of PH . Additionally, these mice show advanced right heart failure, evidenced by increased right ventricular hypertrophy and elevated expression of failure markers like Nppa .

• Methodological approach to study this pathway: Researchers should consider using:

  • Targeted lipidomic profiling for quantifying specific epoxide species

  • Hypoxia-induced PH models with PAFAH2 modulation (genetic or pharmacological)

  • Combined pulmonary and cardiac functional assessments

What pathogenic variants of PAFAH2 have been identified, and how should they be experimentally characterized?

Two particularly significant pathogenic variants of PAFAH2 have been identified in pulmonary arterial hypertension (PAH) patients through whole-exome sequencing:

• Identified variants:

  • p.Arg85Cys (R85C)/c.253C>T

  • p.Gln184Arg (Q184R)/c.551A>G

• Pathogenicity prediction:

  • Both variants demonstrate high Combined Annotation Dependent Depletion (CADD) scores, indicating potential pathogenicity

  • Importantly, these variants are located at sites distinct from the catalytic domains of PAFAH2

• Structural effects:

  • Homology modeling studies revealed that both variants induce conformational changes in the PAF-AH2 protein compared to the native structure

  • These conformational alterations likely contribute to protein instability

• Protein stability:

  • In vitro expression studies showed significantly reduced protein levels of PAF-AH2 p.R85C and p.Q184R variants compared to wild-type PAF-AH2

  • In contrast, a variant at the catalytic site (S236C) did not exhibit reduced protein levels

• Degradation mechanism:

  • Treatment with MG132, a proteasome inhibitor, partially restored protein levels of both R85C and Q184R variants

  • This finding suggests that the ubiquitin-proteasome system mediates the degradation of these variant proteins

• Experimental characterization approach:

  • Site-directed mutagenesis of PAFAH2 expression constructs

  • Protein expression analysis with proteasome inhibition studies

  • Enzymatic activity assays to assess functional consequences

  • Structural modeling to predict conformational changes

What are the most effective strategies for troubleshooting non-specific binding of PAFAH2 antibodies?

When encountering non-specific binding with PAFAH2 antibodies, researchers should implement the following troubleshooting strategies:

• Antibody validation:

  • Confirm antibody specificity using PAFAH2 knockout or knockdown samples as negative controls

  • Validate antibody binding to recombinant PAFAH2 protein

  • Consider testing multiple antibodies targeting different epitopes of PAFAH2

• Blocking optimization:

  • Test different blocking agents: BSA, normal serum from the secondary antibody host species, or commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration in IHC/ICC applications

• Antibody dilution optimization:

  • Perform titration experiments with a range of dilutions (1:100 to 1:5000)

  • For Western blotting, higher dilutions (1:1000-1:5000) often reduce background

  • For IHC/ICC, moderate dilutions (1:100-1:500) typically provide optimal signal-to-noise ratio

• Washing protocol enhancement:

  • Increase wash buffer volume and duration between antibody incubations

  • Add 0.1% Tween-20 to wash buffer to reduce non-specific hydrophobic interactions

  • Implement additional washing steps after secondary antibody incubation

• Pre-adsorption with blocking peptides:

  • For critical experiments, consider pre-adsorbing the antibody with its immunizing peptide

  • Compare staining patterns with and without pre-adsorption to identify specific signals

• Cross-reactivity assessment:

  • Be aware that some PAFAH2 antibodies might cross-react with related family members like PAFAH1B2

  • Perform parallel experiments with antibodies against related proteins to identify potential cross-reactivity

How should experiments be designed to assess PAFAH2 enzymatic activity in various sample types?

Designing rigorous experiments to assess PAFAH2 enzymatic activity requires careful consideration of sample preparation, assay conditions, and appropriate controls:

• Sample preparation protocols:

  • Tissue samples: Homogenize in ice-cold buffer containing protease inhibitors without serine protease inhibitors (which may inhibit PAFAH2)

  • Cell lysates: Use gentle lysis buffers (e.g., NP-40 buffer) to preserve enzymatic activity

  • Subcellular fractionation: Consider separating cytosolic and membrane fractions to localize activity

• Activity assay methodology:

  • Substrate selection: Use PAF or PAF analogs with radiolabeled or fluorescently labeled acetyl groups at the sn-2 position

  • Reaction conditions: Typically perform assays at physiological pH (7.4) and temperature (37°C)

  • Time course analysis: Establish linear range of enzyme activity by sampling at multiple time points

  • Product detection: Quantify hydrolysis products by TLC, HPLC, or specialized colorimetric/fluorometric kits

• Critical controls:

  • Positive control: Recombinant PAFAH2 protein or samples with confirmed high PAFAH2 activity

  • Negative control: Heat-inactivated enzyme or samples from PAFAH2 knockout models

  • Specificity control: Include selective PAFAH2 inhibitors to confirm the specificity of measured activity

  • Background control: Run assays without sample to account for non-enzymatic substrate degradation

• Complementary approaches:

  • Correlate enzymatic activity with protein expression levels determined by Western blotting

  • Consider transacetylase activity assays using lysoplasmalogen or sphingosine as acceptors

  • For complex samples, immunoprecipitate PAFAH2 prior to activity measurements

What are the key considerations for designing in vivo experiments to study PAFAH2 function in pulmonary hypertension models?

Designing robust in vivo experiments to investigate PAFAH2 function in pulmonary hypertension models requires careful attention to multiple experimental parameters:

• Model selection:

  • Hypoxia-induced PH: Chronic hypoxia (10% O₂) for 3-4 weeks has been validated for studying PAFAH2 function

  • Monocrotaline-induced PH: Consider for complementary non-hypoxic model

  • Genetic models: PAFAH2 knockout mice serve as valuable tools for loss-of-function studies

  • Consider generating knock-in models of human PAFAH2 variants (R85C, Q184R) for translational relevance

• Assessment parameters:

  • Hemodynamic measurements: Right ventricular systolic pressure (RVSP) via cardiac catheterization

  • Cardiac remodeling: Right ventricular hypertrophy (Fulton index: RV/[LV+S])

  • Vascular remodeling: Pulmonary arterial wall thickness and muscularization degree

  • Perivascular fibrosis: Masson's trichrome or Sirius red staining

  • Molecular markers: Nppa expression and other heart failure indicators

• Omega-3 epoxide analysis:

  • Targeted lipidomic profiling for quantifying 17,18-EpETE, 19,20-EpDPE, and related metabolites

  • Compare epoxide levels between wild-type and PAFAH2-modified animals under normoxic and hypoxic conditions

• Intervention studies:

  • Rescue experiments with exogenous omega-3 epoxides in PAFAH2 knockout animals

  • Dietary interventions with omega-3 fatty acid supplementation

  • Pharmacological modulation of PAFAH2 activity or stability

• Statistical considerations:

  • Power analysis to determine appropriate sample size (typically 8-12 animals per group for PH studies)

  • Account for sex-specific differences by analyzing male and female animals separately

  • Include time-course analyses to capture disease progression

What methods should be used to quantify PAFAH2 protein expression in clinical samples?

Accurate quantification of PAFAH2 protein expression in clinical samples requires selecting appropriate methodologies based on sample type and research objectives:

• Tissue sample analysis:

  • Immunohistochemistry (IHC-P):

    • Optimal fixation: 10% neutral buffered formalin for 24-48 hours

    • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

    • Primary antibody: Rabbit polyclonal antibodies have been validated for human samples

    • Quantification: Consider digital pathology approaches with positive pixel algorithms

  • Western blotting:

    • Tissue lysis: RIPA buffer with protease inhibitors

    • Loading control: β-actin or GAPDH for normalization

    • Detection: Enhanced chemiluminescence systems with digital imaging

    • Quantification: Densitometric analysis normalized to loading controls

• Liquid biopsy analysis:

  • Magnetic Luminex assay:

    • Validated for serum, plasma, and other biological fluids

    • Requires specialized equipment (Luminex MAGPIX® or similar systems)

    • Offers highly sensitive quantitative measurement

    • Consider multiplex approaches to measure PAFAH2 alongside related biomarkers

  • ELISA:

    • Several PAFAH2 antibodies are validated for ELISA applications

    • Suitable for high-throughput screening of clinical samples

    • Consider sandwich ELISA format for improved specificity

• Cell-type specific analysis:

  • Immunocytochemistry/Immunofluorescence:

    • Validated for cellular localization studies

    • Consider co-staining with cell-type specific markers

    • Confocal microscopy for subcellular localization

  • Flow cytometry:

    • For analysis of PAFAH2 in specific cell populations

    • Requires optimization of fixation and permeabilization protocols

    • Consider intracellular staining protocols for cytosolic PAFAH2

• Considerations for variant detection:

  • Western blotting can distinguish wild-type from variant PAFAH2 proteins based on stability differences

  • Proteasome inhibition with MG132 can help detect unstable variants by preventing their degradation

  • Consider targeted mass spectrometry approaches for direct detection of variant peptides

How should researchers interpret conflicting PAFAH2 expression data across different detection methods?

When faced with conflicting PAFAH2 expression data from different detection methods, researchers should implement a systematic approach to reconcile discrepancies:

• Methodological differences assessment:

  • Consider the detection sensitivity of each method:

    • Western blotting: Good for total protein levels but semi-quantitative

    • IHC/ICC: Provides spatial information but may be less quantitative

    • Luminex/ELISA: Highly quantitative but loses spatial information

  • Evaluate epitope accessibility differences between methods:

    • Fixed tissues may mask certain epitopes

    • Denatured proteins in Western blotting expose different epitopes than native proteins in ELISA

    • Different antibodies target different regions of PAFAH2, potentially yielding different results

• Sample preparation variables:

  • Fixation effects: Overfixation in formalin can reduce epitope detection in IHC

  • Protein extraction efficiency: Different lysis buffers may extract PAFAH2 with varying efficiency

  • Protein degradation: PAFAH2 variants (R85C, Q184R) are known to have reduced stability

  • Proteasomal degradation: Consider whether samples were collected with proteasome inhibitors

• Resolution approach:

  • Triangulate with a third, independent method

  • Test multiple antibodies targeting different PAFAH2 epitopes

  • Include recombinant PAFAH2 protein as a positive control across methods

  • Use PAFAH2 knockout/knockdown samples as negative controls

  • Consider enzyme activity assays as a functional readout to complement expression data

• Data integration strategy:

  • Weight results based on methodological strengths for your specific research question

  • Report discrepancies transparently in publications

  • Consider protein modification or degradation as biological explanations for differences

  • Evaluate whether conflicting data might reflect different PAFAH2 isoforms or variants

What are the best practices for validating novel PAFAH2 variants identified in patient samples?

Validation of novel PAFAH2 variants requires a comprehensive, multi-faceted approach:

• Genetic validation:

  • Confirm variant by bidirectional Sanger sequencing

  • Determine allele frequency in population databases (gnomAD, 1000 Genomes)

  • Perform segregation analysis in families when possible

  • Calculate CADD scores or use other pathogenicity prediction algorithms

• Structural analysis:

  • Generate homology models to predict conformational changes induced by variants

  • Map variants relative to catalytic sites and known functional domains

  • Use molecular dynamics simulations to assess potential effects on protein stability

  • Consider how variants might affect protein-protein interactions

• Expression analysis:

  • Clone wild-type and variant PAFAH2 into expression vectors (e.g., pcDNA)

  • Transfect into relevant cell types (e.g., pulmonary artery cells for PAH research)

  • Quantify protein levels via Western blotting with appropriate controls

  • Assess subcellular localization using immunofluorescence

• Stability assessment:

  • Perform cycloheximide chase assays to measure protein half-life

  • Test proteasome inhibitors (e.g., MG132) to determine degradation mechanisms

  • Consider lysosomal inhibitors to rule out alternative degradation pathways

  • Evaluate ubiquitination status via immunoprecipitation followed by ubiquitin Western blotting

• Functional characterization:

  • Measure enzymatic activity using PAF hydrolysis assays

  • Assess transacetylase activity with appropriate substrates

  • Quantify omega-3 epoxide production capacity in relevant cellular models

  • Evaluate effects on downstream signaling pathways

• Disease model validation:

  • Generate knock-in mouse models of specific variants

  • Challenge with hypoxia or other PH-inducing conditions

  • Assess pulmonary vascular remodeling and right heart function

  • Measure omega-3 epoxide levels in relevant tissues

What specialized methods exist for studying PAFAH2's role in omega-3 fatty acid metabolism?

Investigating PAFAH2's role in omega-3 fatty acid metabolism requires specialized techniques:

• Lipidomic profiling approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for targeted quantification of:

    • 17,18-EpETE (epoxyeicosatetraenoic acid) derived from EPA

    • 19,20-EpDPE (epoxydocosapentaenoic acid) derived from DHA

  • Sample preparation considerations:

    • Rapid tissue harvesting and snap-freezing to prevent ex vivo lipid metabolism

    • Extraction with appropriate organic solvents (e.g., methanol/chloroform mixtures)

    • Addition of antioxidants (e.g., BHT) to prevent oxidation during processing

  • Internal standards:

    • Deuterated omega-3 epoxides as internal standards for accurate quantification

    • Consider stable isotope-labeled precursors for metabolic flux analyses

• Enzyme activity characterization:

  • Recombinant enzyme approach:

    • Express and purify wild-type and variant PAFAH2 proteins

    • Incubate with EPA or DHA substrates under physiological conditions

    • Quantify epoxide formation using LC-MS/MS

  • Cell-based systems:

    • Overexpress or knock down PAFAH2 in relevant cell types

    • Supplement media with EPA or DHA

    • Measure intracellular and secreted epoxide levels

  • Tissue explant cultures:

    • Culture lung tissue slices from wild-type and PAFAH2 knockout mice

    • Incubate with EPA/DHA with or without enzyme inhibitors

    • Analyze epoxide production capacity

• Functional significance assessment:

  • Vascular reactivity studies:

    • Isolated pulmonary artery rings from wild-type and PAFAH2 knockout mice

    • Measure vasodilatory responses to exogenous omega-3 epoxides

    • Assess endothelial-dependent and independent relaxation

  • Cell signaling analyses:

    • Evaluate how omega-3 epoxides affect key signaling pathways in pulmonary arterial cells

    • Measure calcium flux, NO production, or cAMP levels

    • Identify downstream effectors using phosphoproteomic approaches

• Translational methodologies:

  • Human biospecimen analysis:

    • Measure omega-3 epoxides in plasma or bronchoalveolar lavage fluid from PAH patients

    • Correlate with PAFAH2 genotype (wild-type vs. variants)

    • Analyze lung tissue samples when available

  • Ex vivo human lung perfusion models:

    • Test effects of PAFAH2 modulation on pulmonary vascular resistance

    • Measure omega-3 epoxide production in response to EPA/DHA supplementation

What emerging therapeutic strategies might target the PAFAH2 pathway for treating pulmonary hypertension?

Based on current understanding of PAFAH2 biology, several promising therapeutic strategies emerge:

• Direct enzyme stabilization approaches:

  • Small molecule chaperones to stabilize pathogenic PAFAH2 variants (R85C, Q184R)

  • Proteasome modulation to prevent excessive degradation of variant proteins

  • Structure-based drug design targeting variant-specific conformational changes

• Omega-3 epoxide-based interventions:

  • Direct supplementation with purified 17,18-EpETE or 19,20-EpDPE

  • Development of stable epoxide analogs resistant to degradation

  • Dual EPA/DHA supplementation with soluble epoxide hydrolase inhibitors to increase endogenous epoxide levels

  • Nanoparticle delivery systems for targeted pulmonary delivery of epoxides

• Gene therapy approaches:

  • AAV-mediated delivery of wild-type PAFAH2 to pulmonary tissues

  • CRISPR-based correction of pathogenic PAFAH2 variants

  • Antisense oligonucleotides to modulate PAFAH2 expression or splicing

• Combination strategies:

  • PAFAH2 pathway modulation combined with current PAH therapies (PDE5 inhibitors, prostacyclins, ERAs)

  • Multi-target approaches addressing both PAFAH2 and related lipid mediator pathways

  • Personalized approaches based on patient PAFAH2 genotype

• Research directions to enable therapeutic development:

  • High-throughput screening for PAFAH2 stabilizers or activators

  • Pharmacokinetic/pharmacodynamic studies of omega-3 epoxides in pulmonary circulation

  • Development of biomarkers to identify patients most likely to benefit from PAFAH2-targeted therapies

How might advanced imaging techniques enhance our understanding of PAFAH2 localization and function?

Advanced imaging methodologies offer powerful approaches to elucidate PAFAH2 biology:

• Super-resolution microscopy applications:

  • STORM/PALM techniques to visualize PAFAH2 distribution beyond the diffraction limit

  • SIM microscopy for detailed co-localization studies with interaction partners

  • Lattice light-sheet microscopy for dynamic, live-cell imaging of PAFAH2 trafficking

  • Optimization considerations:

    • Antibody selection: Use highly specific antibodies validated for IF applications

    • Fixation protocols: Mild fixation to preserve antigenicity and protein localization

    • Fluorophore selection: Bright, photostable dyes compatible with super-resolution techniques

• Correlative light and electron microscopy (CLEM):

  • Immunogold labeling of PAFAH2 for ultrastructural localization

  • CLEM workflow to correlate functional fluorescence data with ultrastructural context

  • FIB-SEM tomography for 3D ultrastructural analysis of PAFAH2-containing structures

• In vivo imaging approaches:

  • Transgenic reporter systems (e.g., PAFAH2-GFP knock-in mice)

  • Intravital microscopy of pulmonary vasculature in PAFAH2 reporter animals

  • PET imaging with radiolabeled PAFAH2 ligands or substrates

  • Optical imaging using activatable probes responsive to PAFAH2 activity

• Functional imaging techniques:

  • FRET/FLIM sensors to monitor PAFAH2 interactions with binding partners

  • Activity-based probes for visualizing PAFAH2 enzymatic activity in situ

  • Optogenetic approaches to manipulate PAFAH2 activity with spatiotemporal precision

  • Combined calcium imaging and PAFAH2 visualization to correlate with vascular reactivity

• Tissue-scale imaging:

  • Whole-organ imaging using tissue clearing techniques (CLARITY, iDISCO)

  • Light-sheet microscopy of cleared lungs from wild-type and PAFAH2 knockout mice

  • Spatial transcriptomics to correlate PAFAH2 protein localization with gene expression patterns

  • Multi-parametric imaging combining PAFAH2 with cell-type markers and functional readouts