PLA2G4F Antibody

What is PLA2G4F and what are its primary cellular functions?

PLA2G4F (phospholipase A2 group IVF) is a calcium-dependent phospholipase that selectively hydrolyzes glycerophospholipids in the sn-2 position. In humans, the canonical protein has 849 amino acid residues and a molecular weight of approximately 95.1 kDa. PLA2G4F demonstrates higher enzyme activity for phosphatidylethanolamine than phosphatidylcholine, suggesting substrate specificity that differentiates it from other phospholipase family members .

This enzyme plays potential roles in membrane lipid remodeling and biosynthesis of lipid mediators. It is predicted to be involved in several biological processes including glycerophospholipid catabolic processes, arachidonate secretion, cellular response to antibiotics, and prostaglandin biosynthetic processes . The protein is predominantly expressed in myocardium, which suggests tissue-specific functions that may be particularly relevant for cardiovascular research .

What is the subcellular localization of PLA2G4F?

PLA2G4F exhibits multiple subcellular localizations, being found in the cell membrane, mitochondria, and cytoplasm. Additionally, it is predicted to be active in cytosol, ruffle membrane, and vesicles . This diverse distribution suggests that PLA2G4F likely participates in various cellular processes depending on its subcellular context. When designing experiments to study PLA2G4F, researchers should consider this compartmentalization, as it may affect experimental approaches for protein isolation and functional studies .

What are the most common applications for PLA2G4F antibodies?

PLA2G4F antibodies are utilized across several experimental techniques, with Western Blot being the most common application. Other frequently employed methods include ELISA and immunohistochemistry . The table below summarizes the primary applications for commercially available PLA2G4F antibodies and their associated characteristics:

When selecting a PLA2G4F antibody, researchers should prioritize those validated for their specific application of interest, as performance can vary significantly between different experimental contexts .

What are the optimal experimental conditions for using PLA2G4F antibodies in Western blot?

For optimal Western blot detection of PLA2G4F, researchers should consider the following methodological recommendations:

• Sample preparation: Due to PLA2G4F's multiple cellular localizations, use extraction buffers that effectively solubilize membrane, cytoplasmic, and mitochondrial proteins. RIPA buffer with protease inhibitors is often suitable for this purpose.

• Gel percentage: Use 8-10% polyacrylamide gels to effectively resolve the 95.1 kDa PLA2G4F protein.

• Transfer conditions: Employ wet transfer methods with methanol-containing buffers for optimal transfer of this higher molecular weight protein.

• Blocking: 5% non-fat dry milk or BSA in TBST is typically effective.

• Antibody dilution: Optimal dilutions must be determined experimentally but typically range from 1:500 to 1:2000 for primary antibodies .

• Detection system: Both chemiluminescent and fluorescent detection systems are compatible, with the latter offering better quantitative analysis capability.

When troubleshooting Western blots, researchers should verify the expected molecular weight (95.1 kDa) while being aware that post-translational modifications may cause slight variations in migration patterns .

How can researchers validate the specificity of PLA2G4F antibodies?

Validating antibody specificity is crucial for reliable experimental results. For PLA2G4F antibodies, the following validation methods are recommended:

• Positive controls: Use samples with known expression of PLA2G4F, such as myocardial tissue extracts or transfected cell lines overexpressing PLA2G4F .

• Negative controls: Employ samples where PLA2G4F expression is absent or significantly reduced, such as non-transfected control cells or tissue types with minimal expression .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding sites, which should eliminate or significantly reduce the specific signal.

• Knockout/knockdown validation: Use samples from knockout models or siRNA-treated cells to confirm antibody specificity.

• Multiple antibody validation: Compare results using antibodies targeting different epitopes of PLA2G4F to confirm specificity of detection .

These validation steps help ensure that experimental observations are genuinely related to PLA2G4F rather than non-specific interactions or cross-reactivity with other proteins .

What factors affect PLA2G4F antibody cross-reactivity across species?

Cross-species reactivity is an important consideration when selecting PLA2G4F antibodies. The sequence homology of PLA2G4F varies across species, which directly impacts antibody recognition. Current commercial antibodies show confirmed reactivity with human and mouse PLA2G4F, with predicted reactivity to pig, bovine, horse, sheep, and dog .

When evaluating cross-reactivity potential, researchers should consider:

• Epitope conservation: The immunogen sequence used to generate the antibody should be examined for conservation across species. For example, some available antibodies are generated against sequences with 77% identity to mouse orthologs but only 42% to rat orthologs .

• Validation data: Review manufacturer-provided validation data for specific species reactivity.

• Pilot experiments: When working with species not explicitly validated, conduct pilot experiments with appropriate positive and negative controls.

• Alternative epitopes: Consider antibodies targeting more conserved regions of PLA2G4F if cross-species applications are necessary .

Understanding these factors can help researchers select the most appropriate antibody for their experimental model and avoid misleading results due to species-specific differences in antibody recognition .

What are the challenges in detecting specific isoforms of PLA2G4F?

PLA2G4F has up to three reported isoforms, which presents challenges for specific isoform detection . Researchers should consider:

• Epitope location: Determine whether the antibody's epitope is present in all isoforms or is specific to particular variants. Antibodies targeting common regions will detect all isoforms, while those against unique regions may be isoform-specific.

• Molecular weight discrimination: The reported molecular weight of 95.1 kDa corresponds to the canonical isoform. In Western blot applications, isoforms may be distinguished by their different molecular weights, requiring appropriate gel resolution.

• Isoform-specific antibodies: For studies requiring discrimination between isoforms, custom antibodies targeting unique regions may be necessary.

• RT-PCR complementation: Combine antibody-based protein detection with RT-PCR using isoform-specific primers to confirm the presence of specific variants .

These considerations are particularly important when studying tissue-specific expression patterns or functional differences between PLA2G4F isoforms .

What is known about PLA2G4F's role in pathological conditions?

Recent research has begun to elucidate potential connections between PLA2G4F and disease states. A genome-wide association study identified that copy number deletion of PLA2G4F may be associated with increased risk of schizophrenia, suggesting a potential neuropsychiatric connection for this phospholipase .

While direct research on PLA2G4F in disease contexts remains limited, the enzyme's fundamental role in membrane lipid remodeling and lipid mediator biosynthesis suggests potential implications for:

• Cardiovascular pathologies: Given its expression in myocardium, PLA2G4F may participate in cardiac pathophysiology.

• Inflammatory conditions: As a phospholipase involved in arachidonate secretion and prostaglandin biosynthesis, PLA2G4F could influence inflammatory responses.

• Metabolic disorders: The enzyme's role in glycerophospholipid metabolism suggests potential involvement in lipid metabolism disorders .

Researchers investigating PLA2G4F in disease contexts should consider both its enzymatic activity and potential alterations in expression levels across different pathological states .

How does PLA2G4F differ from other phospholipase A2 family members?

PLA2G4F (cytosolic phospholipase A2 zeta) belongs to the Group IV phospholipase A2 family but possesses distinct properties:

• Substrate preference: PLA2G4F shows higher enzymatic activity toward phosphatidylethanolamine compared to phosphatidylcholine, unlike some other family members .

• Cellular localization: While many phospholipases have specific subcellular localizations, PLA2G4F demonstrates distribution across multiple compartments, including the cell membrane, mitochondria, and cytoplasm .

• Tissue expression: PLA2G4F is predominantly expressed in myocardium, suggesting tissue-specific functions that may differ from other family members with broader expression patterns .

• Structural features: Within the gene family, PLA2G4F has unique structural elements that influence its calcium-dependent phospholipid binding and enzymatic activity .

Understanding these differences is crucial when designing experiments to specifically study PLA2G4F rather than related phospholipases, particularly when considering antibody specificity and functional assays .

What are the recommended controls when using PLA2G4F antibodies?

Implementing appropriate controls is essential for reliable interpretation of results when working with PLA2G4F antibodies. Recommended controls include:

• Positive tissue controls: Myocardium samples are ideal positive controls due to known PLA2G4F expression .

• Recombinant protein standards: Purified or recombinant PLA2G4F can serve as positive controls and aid in identifying the correct molecular weight band.

• Primary antibody omission: To assess non-specific binding of secondary antibodies or detection reagents.

• Isotype controls: Use matched isotype control antibodies to evaluate non-specific binding based on antibody class.

• Knockdown/knockout samples: Cells or tissues with reduced or eliminated PLA2G4F expression serve as specificity controls.

• Peptide competition: Pre-incubation of the antibody with immunizing peptide should eliminate specific signals .

• Loading controls: For quantitative analyses, include appropriate loading controls (e.g., GAPDH, β-actin) to normalize expression levels.

Implementing these controls helps distinguish specific from non-specific signals and increases confidence in experimental observations .

What methodological considerations are important for immunohistochemical detection of PLA2G4F?

When performing immunohistochemistry (IHC) with PLA2G4F antibodies, researchers should address several methodological considerations:

• Fixation protocol: Optimize fixation conditions to preserve PLA2G4F epitopes while maintaining tissue morphology. Both paraformaldehyde fixation for frozen sections and formalin fixation for paraffin embedding can be suitable .

• Antigen retrieval: For FFPE tissues, heat-induced epitope retrieval may be necessary to expose PLA2G4F epitopes masked during fixation.

• Blocking parameters: Use appropriate blocking solutions (e.g., serum from the same species as the secondary antibody) to minimize background staining.

• Antibody concentration: Titrate primary antibody concentrations to determine optimal dilutions that maximize specific signal while minimizing background.

• Detection system selection: Choose detection systems based on the required sensitivity, with polymer-based systems often providing better signal-to-noise ratios than traditional ABC methods.

• Co-localization studies: Consider dual immunofluorescence staining to examine PLA2G4F co-localization with subcellular markers for membrane, mitochondria, or cytoplasmic compartments .

These considerations help ensure specific, reproducible detection of PLA2G4F in tissue samples and cellular preparations .