PON2 Antibody

What experimental applications are most reliable for PON2 detection?

PON2 can be reliably detected using various experimental approaches:

These applications provide complementary information about PON2 expression, localization, and interactions. For western blotting, always run positive controls such as HepG2, HeLa, or L02 cells, which show reliable PON2 expression .

How do I distinguish between different PON2 isoforms?

PON2 exists in multiple isoforms that appear as different molecular weight bands on western blots:

• 40 kDa band: Truncated version lacking residues 123-134 (isoform 2)

• 43 kDa band: Full-length canonical protein (isoform 1)

• 55 kDa band: Reported in some tissues (particularly brain, kidney, and testis)

To distinguish these isoforms:

• Use high-resolution SDS-PAGE (10-12% gels)

• Include positive controls with known isoform expression patterns

• Consider using isoform-specific antibodies when available

• Verify with mass spectrometry for definitive isoform identification

What are the key considerations for designing PON2 knockdown experiments?

When designing PON2 knockdown studies:

• Select appropriate cell models: Consider natural PON2 expression levels (HeLa, HepG2, and K562 cells have substantial baseline expression)

• Use validated siRNA/shRNA sequences: Target conserved regions to affect all isoforms

• Include proper controls:

  • Non-targeting control siRNA

  • Rescue experiments with PON2 constructs resistant to siRNA

• Timing considerations: PON2 knockdown alone may induce cell death in certain cancer cell lines without additional stimulation

• Measurement methods: Verify knockdown by both qPCR and western blot

CAUTION: Complete PON2 knockdown may induce spontaneous cell death in some cell types, which could complicate interpretation of results aimed at studying other functions .

How should I optimize western blotting protocols for detecting multiple PON2 isoforms simultaneously?

To successfully detect multiple PON2 isoforms:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors to preserve post-translational modifications

  • Avoid excessive heating (65°C for 5 minutes preferred over 95°C)

• Gel electrophoresis:

  • Use gradient gels (8-15%) for better separation of isoforms

  • Load adequate protein (25-50 μg total protein per lane)

  • Include recombinant PON2 standards when available

• Transfer and detection:

  • Use PVDF membranes (0.45 μm pore size)

  • Optimize transfer conditions: 25V overnight at 4°C works better than rapid transfers

  • Block with 5% non-fat dry milk in TBST

  • Primary antibody incubation at 4°C overnight (1:500-1:1000 dilution)

  • Use highly sensitive ECL detection systems

• Controls:

  • Include both cell types with known PON2 isoform patterns

  • Consider including samples from PON2 knockout cells as negative controls

What approaches can differentiate between PON paralogues (PON1, PON2, PON3) in experimental systems?

Differentiating between PON family members requires careful experimental design:

• Antibody selection:

  • Use highly specific monoclonal antibodies validated against all three PON proteins

  • Verify specificity using recombinant proteins or knockout samples

  • Example: ab192038 shows no cross-reactivity with PON1 or PON3 recombinant proteins

• Expression pattern analysis:

  • PON1: Predominantly in liver and blood

  • PON2: Widely expressed intracellularly across tissues

  • PON3: Primarily in liver and kidney

• Subcellular localization:

  • PON1 and PON3: Secreted, associated with HDL

  • PON2: Intracellular (nuclear envelope, endoplasmic reticulum, mitochondria)

• Functional assays:

  • PON1: Strong paraoxonase activity

  • PON2: Highest lactonase activity, no paraoxonase activity

  • PON3: Intermediate profile

How do I accurately assess PON2 subcellular localization across different cell types?

PON2 subcellular localization varies by cell type and may include the nuclear envelope, endoplasmic reticulum, and mitochondria. For accurate assessment:

• Cell fractionation approach:

  • Use differential centrifugation to isolate subcellular fractions

  • Verify fraction purity with compartment-specific markers:

    • ER: Calnexin or PDI

    • Mitochondria: VDAC or COX IV

    • Nuclear envelope: Lamin B

  • Analyze PON2 distribution by western blot across fractions

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde (avoid methanol which can disrupt membranes)

  • Permeabilize with 0.1% Triton X-100

  • Use co-staining with organelle markers:

    • ER: Calnexin or PDI

    • Mitochondria: MitoTracker or TOM20

    • Nuclear envelope: Lamin B

  • Analyze using confocal microscopy for accurate co-localization assessment

  • Calculate Pearson's correlation coefficient for quantitative co-localization analysis

• Live cell imaging (when possible):

  • Use PON2-GFP fusion proteins for real-time localization studies

  • Co-stain with organelle-specific dyes

  • Monitor dynamic changes in localization under different conditions

How should I interpret variations in PON2 expression between different disease models?

PON2 expression varies significantly in different disease contexts, requiring careful interpretation:

When analyzing conflicting results:

• Consider the specific cell/tissue type being studied

• Account for experimental methodology differences

• Evaluate whether acute vs. chronic conditions were examined

• Analyze whether genetic variants may influence expression patterns

How do I account for post-translational modifications when studying PON2?

PON2 undergoes several post-translational modifications that affect its function and detection:

• Glycosylation:

  • Affects apparent molecular weight

  • May influence antibody recognition

  • Can be assessed using glycosidase treatments prior to western blotting

• Ubiquitination:

  • Documented at positions K29, K144, K156, K159, and K313

  • Affects protein stability and turnover

  • Can be detected using ubiquitination-specific antibodies or MS/MS analysis

• ADP-ribosylation:

  • Identified at position D124

  • May affect lactonase activity

  • Absent in truncated isoform 2

When analyzing PON2 data:

• Consider treating samples with deglycosylating enzymes to normalize apparent molecular weights

• Use phosphatase inhibitors during sample preparation to preserve phosphorylation states

• Consider that PTMs cluster near polymorphic sites (A148G and S311C), potentially affecting function

• For definitive PTM mapping, employ mass spectrometry approaches

What explains discrepancies in PON2 antibody reactivity across different studies?

Several factors contribute to inconsistent PON2 antibody reactivity:

• Epitope specificity:

  • Antibodies targeting different regions may detect different subsets of PON2 isoforms

  • Some epitopes may be masked by post-translational modifications

  • Conformational epitopes may be lost during denaturation

• Tissue/sample preparation variables:

  • Fixation methods significantly impact epitope availability

  • Antigen retrieval methods differ in effectiveness (TE buffer pH 9.0 often superior for IHC)

  • Protein extraction methods may selectively enrich certain isoforms

• PON2 polymorphisms:

  • Common variants (A148G, S311C) may affect antibody binding

  • Population-specific polymorphism frequencies could explain cross-study variations

To address these discrepancies:

• Validate antibody specificity using knockout models or siRNA

• Test multiple antibodies targeting different epitopes

• Standardize sample preparation and antigen retrieval protocols

• Include appropriate positive controls with known PON2 expression patterns

How can I design experiments to investigate PON2's role in oxidative stress protection?

To study PON2's antioxidant functions:

• Cellular models:

  • Compare wild-type, PON2-overexpressing, and PON2-knockdown cells

  • Use cells with endogenous PON2 expression (HepG2, HeLa, endothelial cells)

  • Consider primary cells from PON2 knockout mice as controls

• Oxidative stress induction methods:

  • H₂O₂ treatment (100-500 μM)

  • Glucose-oxygen deprivation (OGD) for ischemia models

  • Oxidized phospholipids exposure

  • Pro-oxidant chemicals (paraquat, rotenone)

• Measurement endpoints:

  • ROS quantification using DCF-DA or MitoSOX

  • Lipid peroxidation markers (MDA, 4-HNE)

  • Mitochondrial membrane potential (TMRM, JC-1)

  • Cell viability and apoptosis markers

• Mechanistic investigations:

  • Assess mitochondrial function (Seahorse assays)

  • Measure Nrf2 nuclear translocation by western blot or immunofluorescence

  • Evaluate GSK-3β phosphorylation status

  • Quantify antioxidant enzyme expression/activity (SOD, catalase)

What experimental approaches can assess PON2's role in bacterial quorum sensing inhibition?

To investigate PON2's role in quorum sensing:

• Enzymatic activity assays:

  • Use synthetic substrates like 3OC12-HSL

  • Measure hydrolysis rates with recombinant PON2

  • Compare wild-type vs. SNP variant activities

• Bacterial biofilm models:

  • Pseudomonas aeruginosa biofilm formation assays

  • Co-culture PON2-expressing cells with bacteria

  • Compare biofilm formation in the presence of PON2-expressing vs. PON2-deficient cells

• Infection models:

  • Compare wild-type and PON2-knockout mouse susceptibility to infection

  • Analyze epithelial cell responses to bacterial components

  • Assess quorum sensing molecule degradation in different cell types

• Analytical approaches:

  • HPLC or LC-MS quantification of quorum sensing molecules

  • Assess virulence factor expression in bacteria exposed to PON2

  • Evaluate epithelial barrier integrity in infection models

How should I investigate the relationship between PON2 expression and cancer cell apoptosis resistance?

For studying PON2's role in cancer cell apoptosis:

• Expression analysis in cancer models:

  • Compare PON2 levels across cancer vs. normal tissue (IHC, western blot)

  • Analyze PON2 expression in relation to cancer progression/staging

  • Examine correlations with patient survival data

• Functional manipulation approaches:

  • Stable overexpression of PON2-GFP or PON2-HA

  • siRNA/shRNA knockdown of PON2

  • CRISPR/Cas9 knockout models

  • Pharmacological inhibition (when available)

• Apoptosis induction protocols:

  • Chemotherapeutic agents (doxorubicin, imatinib)

  • ER stress inducers (thapsigargin, tunicamycin)

  • Extrinsic pathway activators (TRAIL, FasL)

  • Intrinsic pathway activators (staurosporine)

• Measurement endpoints:

  • Caspase activation (caspases 8, 9, and 3)

  • DNA fragmentation (TUNEL assay)

  • Phosphatidylserine externalization (Annexin V)

  • Mitochondrial membrane potential changes

  • CHOP and JNK activation status

• Mechanistic investigations:

  • Mitochondrial ROS production

  • UPR pathway activation

  • C-Jun phosphorylation status

  • IGF-1 signaling pathway components

What are the optimal experimental designs for studying PON2 in neurodegenerative disease models?

For investigating PON2 in neurodegeneration:

• Model systems:

  • Primary neurons from wild-type and PON2-knockout mice

  • Human iPSC-derived neurons with PON2 manipulation

  • Brain tissue from patients with neurodegenerative diseases

  • Animal models of Alzheimer's, Parkinson's, or stroke

• Stress paradigms:

  • Oxygen-glucose deprivation/reoxygenation (OGD/R)

  • Beta-amyloid exposure

  • Glutamate excitotoxicity

  • Inflammatory stimuli (LPS, TNF-α)

• Neuroprotection assessment:

  • Neuronal viability assays

  • Oxidative stress markers

  • Mitochondrial function

  • Synaptic integrity markers

• Signaling pathway analysis:

  • Nrf2 nuclear translocation

  • GSK-3β phosphorylation status

  • Antioxidant response element (ARE) activation

  • CHOP and JNK activation

• In vivo evaluations:

  • Behavioral assessments in PON2-knockout mice

  • Histological analysis of neuronal damage

  • Brain region-specific expression of PON2 isoforms

  • Correlation of PON2 polymorphisms with disease outcomes

How can I evaluate PON2's role in mitochondrial function?

To investigate PON2's mitochondrial activities:

• Mitochondrial isolation and PON2 localization:

  • Differential centrifugation for mitochondrial fraction isolation

  • Proteinase K protection assays to determine submitochondrial localization

  • Immunogold electron microscopy for high-resolution localization

• Functional assessments:

  • Oxygen consumption rate (Seahorse XF analyzer)

  • Mitochondrial membrane potential measurements

  • Mitochondrial ROS production (MitoSOX)

  • ATP generation capacity

• Protein interaction studies:

  • Co-immunoprecipitation with mitochondrial proteins

  • Proximity ligation assays for protein interactions

  • BioID or APEX2 proximity labeling

• Disease model applications:

  • Compare mitochondrial function in cells with varying PON2 expression

  • Assess mitochondrial morphology using confocal microscopy

  • Measure mitochondrial DNA damage

  • Evaluate mitochondrial calcium handling

What approaches help investigate interactions between PON2 and viral proteins?

For studying PON2-viral protein interactions:

• Virus models to consider:

  • HIV-1 (reported interactions with gp160 and Rev)

  • SARS-CoV-2 (interactions with E, M, Nsp4, Nsp6, ORF7a, Nsp7b, Nsp8, ORF9b)

  • Other viral pathogens with oxidative stress components

• Interaction verification methods:

  • Co-immunoprecipitation assays

  • GST pull-down with recombinant proteins

  • FRET or BiFC for direct interaction visualization

  • Mass spectrometry-based interactome analysis

• Functional consequence assessments:

  • Viral replication assays in PON2-manipulated cells

  • Oxidative stress measurements during infection

  • Subcellular localization changes upon infection

  • PON2 enzymatic activity modulation by viral proteins

• Mechanistic investigations:

  • Map interaction domains through truncation mutants

  • Assess post-translational modifications induced by viral interaction

  • Evaluate changes in PON2's protein interaction network during infection

How can researchers effectively study PON2 polymorphisms in relation to disease susceptibility?

To investigate PON2 polymorphism associations with disease:

• Study design considerations:

  • Case-control studies with appropriate sample size calculation

  • Longitudinal cohort studies for disease progression

  • Family-based association studies for specific variants

  • Meta-analyses of existing polymorphism studies

• Genotyping approaches:

  • PCR-RFLP for common variants (A148G, S311C)

  • TaqMan assays for high-throughput genotyping

  • Next-generation sequencing for comprehensive polymorphism analysis

  • Imputation techniques for large-scale studies

• Functional validation methods:

  • Site-directed mutagenesis to create variant recombinant proteins

  • Enzymatic activity assays comparing variant forms

  • Cellular models expressing different variants

  • Structural modeling of variant effects on protein function

• Clinical correlations:

  • Stratify disease outcomes by genotype

  • Evaluate gene-environment interactions

  • Assess multiple PON genes simultaneously (PON1, PON2, PON3)

  • Consider haplotype analysis rather than single SNPs