PLA2G4E Antibody, HRP conjugated

What is PLA2G4E and why is it an important target for antibody-based research?

PLA2G4E (Phospholipase A2 Group IVE), also known as cPLA2-epsilon, is a calcium-dependent N-acyltransferase involved in the biosynthesis of N-acyl ethanolamines (NAEs) in the brain. This enzyme plays critical roles in several biological processes by transferring the sn-1 fatty acyl chain of phosphatidylcholine to the amine group of phosphatidylethanolamine, generating N-acyl phosphatidylethanolamine (NAPE) .

PLA2G4E has significant research interest due to:

• Its involvement in producing bioactive NAEs, including anandamide

• Its role in regulating membrane trafficking requiring modulation of membrane curvature

• Its promotion of tubule formation in clathrin-independent endocytotic trafficking

• Its recently discovered functions in neurodegenerative conditions and skin flap survival

The protein has a calculated molecular weight of 868 amino acids, approximately 99 kDa, and is encoded by gene ID 123745 .

What are the typical applications for HRP-conjugated PLA2G4E antibodies in research protocols?

HRP-conjugated PLA2G4E antibodies are particularly valuable for specific research applications including:

• ELISA assays: Primary application due to direct enzymatic detection without secondary antibodies

• Multiplexed immunoassays: Particularly in cytometric bead arrays where different antibodies against multiple targets can be used simultaneously

• Quantitative western blotting: Providing enhanced sensitivity and direct detection capability

• Immunohistochemistry: Though less common than unconjugated antibodies for this application

The HRP conjugation enables direct enzymatic visualization through substrate conversion, eliminating the need for secondary antibody incubation steps, which offers significant time advantages and potentially reduces background in certain experimental settings .

How should sample preparation protocols be modified when using HRP-conjugated PLA2G4E antibodies versus unconjugated versions?

When using HRP-conjugated PLA2G4E antibodies, several methodological adaptations are critical:

Additionally, when using these antibodies for cell staining, researchers should be aware that permeabilization steps may need optimization to preserve both antibody binding capability and HRP activity .

How can I validate the specificity of my HRP-conjugated PLA2G4E antibody in my experimental system?

A comprehensive validation strategy for HRP-conjugated PLA2G4E antibodies should include:

• Positive control tissues/cells: Western blot analysis using validated positive controls such as SH-SY5Y cells, A431 cells, K-562 cells, or NIH/3T3 cells, which have been confirmed to express PLA2G4E

• Knockout/knockdown validation: Using shRNA or CRISPR-based knockdown/knockout models, as demonstrated in studies where PLA2G4E expression was decreased using AAV-Pla2g4e shRNA

• Peptide competition assay: Pre-incubation of the antibody with recombinant PLA2G4E protein (such as the immunogen PLA2G4E fusion protein Ag12808) should abolish specific signal

• Cross-reactivity assessment: Test across multiple species if relevant to your research; current PLA2G4E antibodies have been validated with human, mouse, and rat samples

• Molecular weight verification: Confirm signal appears at the expected molecular weight (~98-99 kDa) under appropriate reducing or non-reducing conditions

• Subcellular localization: Immunofluorescence patterns should match known distribution patterns of PLA2G4E, with particular attention to lysosomal colocalization as demonstrated by p-PLA2G4E colocalization with LAMP1

What methodological considerations are critical when using HRP-conjugated PLA2G4E antibodies in multiplexed assays?

When integrating HRP-conjugated PLA2G4E antibodies into multiplexed systems, researchers should consider these methodological aspects:

• Avoiding spectral overlap: When designing multiplex panels, choose fluorophores or chromogens that have distinct spectral properties from the HRP substrate products

• Antibody cross-reactivity: In cytometric bead arrays, validate that matched antibody pairs (such as the 83645-4-PBS capture and 83645-6-PBS detection system) do not cross-react with other targets in your panel

• Sequential detection strategies: For complex tissue staining, consider sequential rather than simultaneous application of different antibodies

• Quantitative standardization: Include appropriate calibration standards, particularly when quantifying PLA2G4E alongside other targets

• Optimization of substrate concentration: For each assay, determine optimal HRP substrate concentration to avoid signal saturation while maintaining sensitivity

• Data normalization strategies: Develop robust methodologies for normalizing signals across different antibody-target combinations

How can I investigate the role of PLA2G4E in lysosomal membrane permeabilization and necroptosis pathways?

Recent research has established PLA2G4E's role in lysosomal membrane permeabilization (LMP) and necroptosis. To investigate these processes, consider the following experimental approach:

• Lysosomal integrity assessment: Use CTSD (Cathepsin D) diffusion patterns as a marker for lysosomal membrane permeabilization. In functional lysosomes, CTSD appears as punctate staining, while diffuse patterns indicate LMP

• Phosphorylation state analysis: Analyze p-PLA2G4E levels using phospho-specific antibodies to evaluate activation state, as elevated phosphorylation correlates with increased activity

• Colocalization studies: Perform dual immunofluorescence with lysosomal markers (LAMP1) and PLA2G4E antibodies to assess recruitment to lysosomes under stress conditions

• Functional modulation: Use targeted approaches such as:

  • AAV-mediated knockdown (AAV-Pla2g4e shRNA)

  • miRNA-based regulation (Mir504-5p) which has been shown to inhibit PLA2G4E

  • Measure outcomes on cell survival, necroptosis markers, and lysosomal function

• Biochemical activity assays: Utilize ELISA-based methods to quantify PLA2G4E enzyme activity before and after experimental interventions

• Lipidomic profiling: Analyze changes in lipid composition, particularly lysophospholipids and ceramides, which influence lysosomal membrane integrity

What are common issues encountered when using HRP-conjugated PLA2G4E antibodies in Western blotting, and how can these be resolved?

For optimal results, Western blot protocols should be adapted for specific sample types and adjusted based on observed expression levels. The recommended dilution range of 1:1000-1:8000 should be titrated for each experimental system .

How does conjugation to HRP affect PLA2G4E antibody performance in immunohistochemical applications?

HRP conjugation introduces several important considerations for immunohistochemical detection of PLA2G4E:

• Direct detection advantages:

  • Elimination of secondary antibody cross-reactivity issues

  • Reduced background from endogenous immunoglobulins

  • Simplified, shorter protocols with fewer incubation steps

• Sensitivity considerations:

  • Potential reduction in signal amplification compared to two-step detection methods

  • May require higher primary antibody concentrations than unconjugated formats

  • Critical to optimize substrate development time to balance sensitivity and specificity

• Antigen retrieval optimization:

  • For PLA2G4E detection in tissues, TE buffer pH 9.0 is recommended based on validation data

  • Alternative antigen retrieval with citrate buffer pH 6.0 may be employed if needed

• Tissue compatibility:

  • Successfully validated in human skeletal muscle and heart tissues

  • When testing novel tissues, comparison with known positive tissues is essential

• Endogenous peroxidase management:

  • More critical for HRP-conjugated antibodies than unconjugated formats

  • Thorough blocking with hydrogen peroxide (0.3% for 10-15 minutes) before antibody application is recommended

What factors should be considered when comparing data between different PLA2G4E antibody clones and detection methods?

When comparing results obtained using different PLA2G4E antibodies or detection methods, researchers should consider:

• Epitope differences: Different antibodies recognize distinct regions of PLA2G4E:

  • Some target AA 604-868 region

  • Others target AA 751-800 or the C-terminal region

  • These differences can affect detection of specific isoforms or post-translationally modified forms

• Antibody class variations:

  • Polyclonal antibodies (like 29990-1-AP) offer broader epitope recognition

  • Recombinant monoclonal antibodies (like 83645-4-PBS) provide higher specificity and batch consistency

  • These differences influence sensitivity and specificity profiles

• Detection method sensitivity hierarchy:

  • Enhanced chemiluminescence detection of HRP typically offers higher sensitivity than colorimetric detection

  • Fluorescent secondary antibodies may provide superior signal-to-noise in certain applications

  • These differences must be accounted for in quantitative comparisons

• Cross-platform normalization:

  • When comparing ELISA, Western blot, and immunohistochemistry data, use consistent positive controls

  • Consider using recombinant standards across platforms when possible

  • Develop normalization strategies based on validated controls

• Clone-specific optimization:

  • Each antibody may require different blocking strategies and dilutions

  • HRP-conjugated versions may have distinct optimal conditions from unconjugated counterparts

How can PLA2G4E antibodies be utilized to study its role in neurodegenerative disease models?

Recent research has identified PLA2G4E as a potential therapeutic target in Alzheimer's disease and other neurodegenerative conditions. When designing experiments to study this connection:

• Gene expression analysis:

  • Measure PLA2G4E levels in brain tissues from AD models and controls

  • Research has shown significant decreases in PLA2G4E in late-stage AD patients compared to controls

• Functional restoration studies:

  • Use viral vectors for overexpression of PLA2G4E in hippocampal neurons

  • Assess cognitive function before and after intervention

  • Studies have demonstrated that AAV-mediated PLA2G4E overexpression restored cognitive deficits in elderly APP/PS1 mice

• Mechanistic investigations:

  • Examine dendritic spine density in relation to PLA2G4E expression

  • Correlate PLA2G4E levels with synaptic plasticity markers

  • Overexpression of PLA2G4E has been shown to increase dendritic spine development

• Antibody-based techniques for validation:

  • Use Western blotting to confirm overexpression or knockdown

  • Employ immunohistochemistry to localize PLA2G4E in brain regions affected by neurodegeneration

  • Combine with behavioral assessments to correlate protein levels with functional outcomes

• Selective inhibition studies:

  • Evaluate the impact of PLA2G4E on activity-dependent signaling in primary neuronal cultures

  • Design experiments that distinguish between effects on amyloid/tau pathology versus cognitive function

What experimental approaches can be used to investigate PLA2G4E's role in skin flap survival and wound healing?

Recent studies have identified PLA2G4E as a regulator of necroptosis and tissue survival in skin flaps. To explore this function:

• Ischemia-reperfusion models:

  • Create random-pattern skin flaps in appropriate animal models

  • Monitor tissue viability in relation to PLA2G4E expression and activity

• Genetic modulation approaches:

  • Use AAV-Pla2g4e shRNA for knockdown studies

  • Consider miRNA-based regulation with Mir504-5p, which has been shown to inhibit PLA2G4E

  • Measure outcomes on tissue survival and necrosis patterns

• Lysosomal integrity assessment:

  • Analyze lysosomal membrane permeabilization (LMP) using markers like CTSD diffusion patterns

  • Correlate PLA2G4E activity with lysosomal integrity in ischemic tissues

• Lipid composition analysis:

  • Perform LC-MS-based lipidomics to analyze changes in membrane lipid composition

  • Focus on lysophospholipids and ceramides which influence lysosomal membrane permeability

• Phosphorylation state analysis:

  • Use immunoblotting and immunofluorescence to detect phosphorylated PLA2G4E

  • Correlate activation state with tissue survival outcomes

• Therapeutic intervention studies:

  • Design experiments testing inhibition of PLA2G4E as a potential therapeutic approach

  • Measure vascular parameters, cell death markers, and tissue viability outcomes

How can multiplexed antibody approaches be optimized when studying PLA2G4E in relation to other phospholipases?

When investigating PLA2G4E alongside other phospholipases (such as PLA2G4A or PLA2G2A), consider these methodological refinements:

• Antibody panel design:

  • Select antibodies with validated cross-reactivity profiles

  • Consider using matched antibody pairs like 83645-4-PBS (capture) and 83645-6-PBS (detection) for PLA2G4E

  • For PLA2G4A, validated antibodies such as AF6659 have established specificity profiles

• Control strategies:

  • Include phospholipase family members as specificity controls

  • Verify cross-reactivity between different PLA2 antibodies, particularly when they target conserved domains

  • Use recombinant proteins of each phospholipase as standards

• Multiplex platform selection:

  • For protein quantification, cytometric bead arrays offer superior multiplexing capability

  • For tissue analysis, sequential immunofluorescence with spectral unmixing allows detection of multiple targets

  • For activity assays, consider substrate-specific approaches that distinguish between different phospholipases

• Sample preparation considerations:

  • Different phospholipases may require distinct extraction methods for optimal recovery

  • When analyzing multiple family members, verify that extraction conditions are compatible with all targets

  • Consider subcellular fractionation to enrich for specific compartments (cytosolic versus membrane-associated)

• Data normalization and analysis:

  • Develop consistent approaches for normalizing signals across different antibody-target combinations

  • Consider ratiometric analysis of different phospholipase family members

  • Use appropriate statistical methods for comparing patterns across experimental conditions