PLA2G4E Antibody, Biotin conjugated

What is PLA2G4E and why is it significant in research?

PLA2G4E (Phospholipase A2, Group IVE) is a calcium-dependent phospholipase A2 that selectively hydrolyzes glycerophospholipids at the sn-2 position. Its significance lies in its role in several biological processes including lysosomal membrane permeabilization (LMP), necroptosis, and N-acyl phosphatidylethanolamine (NAPE) synthesis. Research has shown that PLA2G4E can function as a calcium-dependent N-acyltransferase (Ca-NAT) in mouse brain, making it relevant to studies involving lipid metabolism and cell death pathways . Methodologically, researchers often focus on its calcium-dependent enzymatic activity when designing experiments to understand its function in various cellular contexts.

What are the optimal storage conditions for PLA2G4E antibody?

For maximum stability and activity retention, PLA2G4E antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity and performance . For practical laboratory management, it is recommended to aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles. The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

How specific is the PLA2G4E antibody for human samples versus other species?

The commercial PLA2G4E antibody described in the search results is specifically reactive to human samples and has been raised against recombinant human cytosolic phospholipase A2 epsilon protein (amino acids 604-868) . While cross-reactivity with other species has not been extensively documented in these results, researchers should be aware that antibodies may exhibit differential binding affinities across species due to sequence homology variations. When designing experiments with other species, validation tests should be performed first to confirm antibody specificity and performance before proceeding with full experimental protocols.

Besides ELISA, what other applications might be suitable for biotin-conjugated PLA2G4E antibody?

While the product information indicates ELISA as the tested application , biotin-conjugated antibodies generally offer versatility for various additional techniques. Methodologically, researchers could explore:

• Immunohistochemistry (IHC) - utilizing biotinylated antibodies with avidin-biotin complex (ABC) methods for tissue section analysis

• Immunoprecipitation coupled with mass spectrometry - leveraging the high-affinity biotin-streptavidin interaction

• Flow cytometry - particularly when coupled with fluorescently labeled streptavidin

• Pull-down assays - for protein-protein interaction studies

For any application beyond ELISA, researchers should conduct preliminary optimization experiments to determine appropriate dilutions and conditions for the specific application .

How can I optimize the use of PLA2G4E antibody in co-localization studies with lysosomal markers?

Based on research findings showing PLA2G4E's association with lysosomes, co-localization studies can be strategically designed. For optimal results, researchers should:

• Use LAMP1 as a validated lysosomal marker, which has been successfully employed in previous studies examining PLA2G4E localization

• Implement confocal microscopy with appropriate filter settings to distinguish between the biotin-conjugated PLA2G4E antibody (visualized with fluorescent streptavidin) and lysosomal markers

• Consider fixation methods carefully - paraformaldehyde fixation (4%) for 10-15 minutes is typically suitable for preserving both protein localization and lysosomal structure

• Include controls for antibody specificity, such as competitive blocking with the immunizing peptide and no-primary-antibody controls

Research has demonstrated increased co-localization of phosphorylated PLA2G4E with LAMP1 in injured tissues, suggesting activation at the lysosomal membrane .

What is the relationship between PLA2G4E activity and lysosomal membrane permeabilization?

PLA2G4E has been identified as a key regulator of lysosomal membrane permeabilization (LMP) in the context of ischemic injury. The mechanism involves:

• Increased phosphorylation and activation of PLA2G4E in ischemic tissues

• Enzymatic cleavage of the sn-2 position of glycerophospholipids, releasing arachidonic acid and lysophospholipids

• Accumulation of lysophospholipids (LPC and LPE) in lysosomal membranes, increasing membrane permeability

• Subsequent release of lysosomal contents into the cytoplasm, triggering necroptosis

Research has shown that inhibition of PLA2G4E through shRNA or miR-504-5p reduces LMP and subsequent necroptosis, promoting cell survival in ischemic conditions . This relationship suggests PLA2G4E as a potential therapeutic target in conditions involving ischemic injury and cell death.

How does calcium regulate PLA2G4E activity and what implications does this have for experimental design?

PLA2G4E is a calcium-dependent enzyme, with implications for experimental design:

• Calcium enhances the enzymatic activity of PLA2G4E, as demonstrated by increased FP-rhodamine labeling in the presence of CaCl₂

• The N-acyltransferase (Ca-NAT) activity of PLA2G4E is blocked by EDTA, confirming calcium dependency

• Ionomycin-induced calcium influx promotes PLA2G4E-mediated production of NAPEs, GP-NAEs, and NAEs

For experimental design, researchers should:

• Include EDTA controls to confirm calcium dependency of observed effects

• Consider calcium concentration in buffers (typically 1-2 mM CaCl₂)

• Explore calcium ionophores like ionomycin as tools to activate PLA2G4E in cellular assays

• Design time-course experiments to capture both immediate and delayed calcium-dependent responses

What are common issues in detecting PLA2G4E activity and how can they be addressed?

Detection of PLA2G4E activity can be challenging due to several factors:

• Low abundance in native tissues - activity-based protein profiling (ABPP) with FP-biotin probe and avidin enrichment followed by LC-MS/MS analysis may be necessary for detection in complex samples

• Potential overlap with other phospholipase activities - using specific inhibitors or genetic knockdown approaches helps isolate PLA2G4E-specific activity

• Calcium dependency - ensuring appropriate calcium concentrations in assay buffers is critical; include both calcium-containing and EDTA-containing conditions as controls

• Cell type specificity - PLA2G4E activity may vary significantly between cell types, with research showing activity in neurons but not glia

Methodologically, researchers can address these issues by:

• Employing recombinant PLA2G4E expression systems for positive controls

• Using site-directed mutagenesis (e.g., S420A mutation) to create catalytically inactive controls

• Implementing metabolic labeling with isotope-labeled substrates for enhanced sensitivity

• Validating antibody specificity through immunodepletion experiments

How can I distinguish between phospholipase activity and N-acyltransferase activity when studying PLA2G4E?

Distinguishing between these activities requires careful experimental design:

• Substrate selection - phospholipase activity typically uses phosphatidylcholine substrates, while N-acyltransferase activity utilizes phosphatidylethanolamine and fatty acid substrates

• Product analysis - phospholipase activity generates free fatty acids and lysophospholipids, while N-acyltransferase activity produces N-acyl phosphatidylethanolamines (NAPEs)

• Targeted lipidomic analysis - LC-MS/MS can be used to specifically identify and quantify the distinct lipid products of each activity

• Metabolic labeling - using ¹³C-isotopically labeled fatty acids (e.g., ¹³C₁₆-palmitic acid) enables tracking newly synthesized products in response to stimuli

Research has shown that PLA2G4E demonstrates robust Ca-NAT activity despite having relatively low phospholipase activity, suggesting functional specialization .

What is the potential role of PLA2G4E in therapeutic applications for ischemic tissue damage?

Research indicates several promising therapeutic applications related to PLA2G4E:

• Inhibition of PLA2G4E through AAV-delivered shRNA significantly reduces lysosomal membrane permeabilization and necroptosis in ischemic skin flaps

• MicroRNA-based approaches, specifically miR-504-5p, have demonstrated inhibitory effects on PLA2G4E expression and subsequent reduction in ischemia-induced cell death

• The relationship between PLA2G4E and necroptosis suggests potential applications in conditions beyond skin flaps, including stroke, myocardial infarction, and transplantation

For research methodologies exploring these applications, investigators should consider:

• In vivo models of ischemia-reperfusion injury

• Quantification of tissue survival using appropriate viability markers

• Assessment of lysosomal integrity through cathepsin release assays

• Combinatorial approaches targeting multiple steps in the LMP-necroptosis pathway

How can metabolic labeling techniques enhance the study of PLA2G4E's role in NAPE and endocannabinoid biosynthesis?

Metabolic labeling offers powerful insights into PLA2G4E function:

• ¹³C-labeled fatty acid precursors (like ¹³C₁₆-palmitic acid) allow time-resolved tracking of newly synthesized lipids

• Combined with calcium mobilization (e.g., ionomycin treatment), this approach can isolate calcium-dependent synthesis of NAPEs, GP-NAEs, and NAEs

• Comparing wild-type PLA2G4E with catalytically inactive mutants (S420A) provides specificity controls

• Mass spectrometry analysis of labeled products enables quantification of synthetic rates and pathway flux

This methodology reveals that PLA2G4E catalyzes the production of NAPEs, which are precursors to bioactive NAEs including anandamide. The research implications extend to endocannabinoid signaling, pain regulation, appetite control, and other physiological processes mediated by NAEs .