pla2g4a Antibody

What is PLA2G4A and why is it an important target for antibody-based detection in research?

PLA2G4A, also known as cytosolic phospholipase A2 (cPLA2), is a highly conserved enzyme present in the cytosol of resting cells that undergoes calcium-directed translocation to perinuclear membranes upon activation. It plays a major role in membrane lipid remodeling and biosynthesis of lipid mediators of the inflammatory response . The enzyme is widely expressed in various cell types including endothelium, smooth muscle, macrophages, neutrophils, fibroblasts, mast cells, and platelets, making it a critical target for understanding inflammation and various disease processes . PLA2G4A antibodies are essential tools for investigating this protein's cellular distribution, expression levels, and functional changes in various pathological conditions including cancer, viral infections, and neuropsychiatric disorders .

What molecular characteristics should researchers consider when selecting a PLA2G4A antibody?

When selecting a PLA2G4A antibody, researchers should consider several key molecular characteristics:

The discrepancy between calculated (85 kDa) and observed (100-110 kDa) molecular weights is a consistent finding and should not be considered an error in detection . This difference is likely due to post-translational modifications such as phosphorylation, which affect the protein's migration on SDS-PAGE gels. When validating antibody specificity, this consistent migration pattern at 100-110 kDa in Western blots can serve as a characteristic feature of proper PLA2G4A detection .

How do I determine the appropriate PLA2G4A antibody for my specific experimental application?

Selection of the appropriate PLA2G4A antibody requires consideration of multiple factors aligned with your experimental design:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, ELISA, etc.). For example, antibody 28924-1-AP has been validated for WB (1:1000-1:4000 dilution) and IHC (1:50-1:500 dilution) .

• Species reactivity: Confirm cross-reactivity with your experimental species. Many PLA2G4A antibodies react with human samples, while some also recognize mouse and rat PLA2G4A .

• Clonality considerations:

  • Polyclonal antibodies (like ABIN571180) offer broader epitope recognition but may have batch-to-batch variation .

  • Monoclonal antibodies provide consistent specificity but may be more sensitive to epitope masking.

• Epitope location relevance: Select antibodies targeting regions of interest:

  • N-terminal domain (AA 1-178) antibodies for studying CaLB domain function

  • Internal region antibodies for general detection

  • Phospho-specific antibodies (pSer505) when studying enzyme activation

• Validation stringency: Prioritize antibodies validated through multiple methods, including knockout/knockdown verification, which provides the strongest evidence of specificity .

For mechanistic studies involving PLA2G4A inhibition, such as in viral infection or cancer research, antibodies that have been validated in functional assays with inhibitors like arachidonyl trifluoromethyl ketone (AACOCF3) are particularly valuable .

What are the optimized protocols for detecting PLA2G4A using Western blotting?

Successful Western blotting for PLA2G4A requires attention to several critical parameters:

Sample preparation:

• Use lysis buffers containing protease inhibitors to prevent degradation

• Include phosphatase inhibitors to preserve phosphorylation states

• Load 20-50 μg of total protein per lane for cell lysates

SDS-PAGE conditions:

• Use 7.5-10% gels for optimal resolution of the 100-110 kDa PLA2G4A band

• Include positive control lysates such as HeLa cells, which consistently express PLA2G4A

Transfer and detection:

• Transfer proteins to PVDF membrane (nitrocellulose is also acceptable)

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

• Primary antibody dilution: 1:1000-1:4000 (optimize for each antibody)

• Incubate with primary antibody overnight at 4°C

• Secondary antibody: HRP-conjugated anti-species IgG (1:5000-1:10000)

• Visualization: Enhanced chemiluminescence systems

Critical considerations:

• Expect the PLA2G4A band at 100-110 kDa, not at the calculated 85 kDa

• Include molecular weight markers spanning 75-120 kDa range

• Strip and reprobe with antibodies against housekeeping proteins (β-actin, GAPDH) as loading controls

• For phosphorylation studies, consider dual probing with total and phospho-specific PLA2G4A antibodies

When interpreting Western blot results, note that PLA2G4A expression can be induced in select cell types, including endothelium, smooth muscle, macrophages, neutrophils, fibroblasts, mast cells, and platelets . Tumor cells often serve as good sources for cPLA2 alpha activity and expression .

How should samples be prepared for optimal PLA2G4A detection in immunohistochemistry?

Successful immunohistochemical detection of PLA2G4A requires specific sample preparation techniques:

Tissue processing and preparation:

• Fix tissues in 10% neutral buffered formalin (24-48 hours)

• Process and embed in paraffin following standard protocols

• Section tissues at 4-5 μm thickness

• Mount sections on positively charged slides

Antigen retrieval (critical step):

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

• Heat-induced epitope retrieval: Pressure cooker or microwave methods

• Retrieval duration: 15-20 minutes at high pressure/temperature

Staining protocol:

• Block endogenous peroxidase (3% H₂O₂, 10 minutes)

• Protein blocking (5-10% normal serum, 30 minutes)

• Primary antibody incubation:

  • Dilution: 1:50-1:500 (optimize for each antibody and tissue)

  • Incubation: Overnight at 4°C or 1 hour at room temperature

• Secondary detection: HRP-polymer detection system

• Visualization: DAB chromogen (3-5 minutes)

• Counterstain: Hematoxylin (30-60 seconds)

Controls and validation:

• Positive tissue control: Human colon cancer tissue has been validated for PLA2G4A detection

• Negative controls: Omit primary antibody on serial sections

• Consider dual staining with cell-type specific markers for colocalization studies

For researchers studying PLA2G4A in colorectal cancer, note that expression patterns may differ between right-sided CRC (RSCRC) and left-sided CRC (LSCRC), with potential prognostic implications .

What control experiments are essential when validating PLA2G4A antibody specificity?

Rigorous validation of PLA2G4A antibody specificity requires multiple complementary approaches:

1. Molecular and genetic controls:

• Knockdown/knockout validation: Use siRNA, shRNA, or CRISPR-Cas9 to reduce PLA2G4A expression

  • Quantify signal reduction through imaging or densitometry

  • Compare with non-targeting controls

  • Expected outcome: Proportional decrease in signal intensity

• Overexpression controls: Compare wild-type cells to those overexpressing PLA2G4A

  • Use tagged constructs (GFP, FLAG, etc.) for dual detection

  • Expected outcome: Increased signal at the appropriate molecular weight

2. Biochemical controls:

• Peptide competition assay: Pre-incubate antibody with immunizing peptide

  • Titrate peptide concentrations (1:1, 5:1, 10:1 peptide:antibody molar ratio)

  • Expected outcome: Dose-dependent reduction in specific signal

• Phosphatase treatment: For phospho-specific antibodies

  • Treat duplicate samples with lambda phosphatase

  • Expected outcome: Loss of signal with phospho-specific antibodies, no change with total PLA2G4A antibodies

3. Multi-antibody validation approach:

• Independent antibody comparison: Use antibodies targeting different PLA2G4A epitopes

  • Compare staining patterns between antibodies targeting N-terminal, internal, and C-terminal regions

  • Expected outcome: Consistent localization patterns and expression levels

4. Sample type controls:

• Known positive samples: HeLa cells (human) and NIH-3T3 cells (mouse) are validated positive controls

• Known negative samples: Cell lines with confirmed low/no PLA2G4A expression

• Tissue-specific expression: Compare with known expression patterns from RNA-seq or proteomics datasets

How can PLA2G4A antibodies be utilized to investigate its role in colorectal cancer progression?

PLA2G4A antibodies enable sophisticated investigation of its role in colorectal cancer (CRC) through several methodological approaches:

Expression profiling in primary tumors:

• Use immunohistochemistry to compare PLA2G4A levels between:

  • Normal colonic mucosa

  • Right-sided vs. left-sided CRC tumors

  • Different histological grades and stages

• Develop scoring systems based on staining intensity and percentage of positive cells

• Correlate expression with clinical parameters and patient survival

Tumor microenvironment studies:

• Use multiplex immunofluorescence to co-localize PLA2G4A with:

  • Immune cell markers (particularly γδ T cells)

  • CD39 expression on tumor-infiltrating lymphocytes

  • Markers of regulatory T cells

This approach has revealed that PLA2G4A expression in RSCRC educates γδ T cells into CD39+ γδ Tregs, creating an immunosuppressive microenvironment that promotes tumor progression and metastasis .

Functional studies in CRC cell lines:

• Create PLA2G4A overexpression and knockdown models in CRC cell lines

• Use antibodies to verify manipulation efficiency

• Study effects on:

  • Cell proliferation and migration

  • Immune cell interaction in co-culture systems

  • Colony formation and tumor spheroid development

Experimental evidence shows that overexpression of Pla2g4a in CT26 cells induced CD39+ γδ Tregs and inhibited antitumor immune responses, while silencing Pla2g4a reduced CD39 expression on γδ T cells and rescued their function .

In vivo validation with orthotopic models:

• Establish orthotopic CRC models with modified PLA2G4A expression

• Use antibodies to track PLA2G4A in tumor tissues

• Correlate with:

  • Tumor weight and metastasis

  • CD39 expression on tumor-infiltrating γδ T cells

  • Histopathological features of invasion

These comprehensive approaches have established PLA2G4A as a critical regulator of tumor-immune interactions in CRC, with potential as both a prognostic marker and therapeutic target .

What methodologies can reveal PLA2G4A's role in viral infections using antibody-based detection?

Antibody-based methodologies have been instrumental in uncovering PLA2G4A's critical role in viral infections, particularly Hepatitis C virus (HCV):

Subcellular localization during infection:

• Employ immunofluorescence microscopy to track PLA2G4A translocation:

  • In uninfected cells: Primarily cytosolic

  • During viral infection: Translocates to perinuclear membranes, ER, and Golgi

• Co-staining with viral proteins (e.g., HCV core) to identify colocalization

• Live-cell imaging with fluorescently tagged antibodies to monitor dynamic changes

Virus assembly investigation:

• Use PLA2G4A antibodies for Western blotting to:

  • Measure expression levels during different stages of viral infection

  • Detect phosphorylation status (activated form)

  • Correlate with viral protein production timelines

• Employ immunoprecipitation to identify viral or host protein interactions:

  • Pull down PLA2G4A and probe for viral proteins

  • Identify host factors in the complex

Functional inhibition studies:

• Combine antibody detection with PLA2G4A pharmacological inhibition:

  • Treat infected cells with arachidonyl trifluoromethyl ketone (AACOCF3)

  • Track changes in viral assembly using antibodies

  • Quantify viral core protein abundance at lipid droplets

Research has shown that inhibition of PLA2G4A activity reduces core protein abundance at lipid droplets, impairs core envelopment, and results in secretion of aberrant particles that are 100-fold less infectious . Remarkably, exogenous addition of arachidonic acid (the cleavage product of PLA2G4A-catalyzed lipolysis) restores infectivity, highlighting the specific requirement for this lipid product .

Viral particle composition analysis:

• Purify viral particles from control and PLA2G4A-inhibited cells

• Use antibodies to analyze protein composition of virions

• Correlate compositional changes with infectivity

These methodologies have revealed previously unrecognized parallels in the assembly pathways of HCV and related viruses like Dengue, defining PLA2G4A-dependent lipolysis as a crucial prerequisite for production of highly infectious viral progeny .

How are PLA2G4A antibodies employed in leukemia research for biomarker and target validation?

PLA2G4A antibodies play critical roles in leukemia research, particularly in acute myeloid leukemia (AML) with MLL rearrangements:

Expression profiling in patient samples:

• Use immunohistochemistry and Western blotting to:

  • Compare PLA2G4A levels between AML subtypes

  • Correlate expression with genetic alterations (MLL rearrangements)

  • Analyze expression in relation to HOXA9 and MEIS1 status

Research has demonstrated that PLA2G4A is significantly higher expressed in AML patients with MLL rearrangement compared to those without, making it a potential independent prognostic marker .

Functional genomics validation:

• Employ CRISPR-screen approaches targeting PLA2G4A:

  • Use fluorescent genetic barcoding (FGB) vectors expressing sgRNAs

  • Track competitive growth behavior of PLA2G4A-modified cells

  • Validate knockdown efficiency with antibodies

  • Correlate with functional outcomes

A multiplex CRISPR-screen identified PLA2G4A as a prognostic marker and potential therapeutic target for HOXA9 and MEIS1 dependent AML .

Pharmacological inhibition studies:

• Combine antibody detection with PLA2G4A inhibitor treatment:

  • Treat AML cells with arachidonyl trifluoromethyl ketone (AACOCF3)

  • Monitor changes in cell viability and proliferation

  • Track colony formation in methylcellulose

  • Correlate outcomes with PLA2G4A expression levels

Experimental evidence shows that pharmacologic inhibition of PLA2G4A with AACOCF3 accelerates the loss of HOXA9-MEIS1 (H9M) cells in bulk cultures and reduces colony numbers and sizes in methylcellulose .

Mechanistic investigations:

• Use antibodies to elucidate signaling pathways:

  • Analyze PLA2G4A activation in response to various stimuli

  • Study downstream effectors of PLA2G4A activity

  • Investigate connections to leukemic stem cell maintenance

These comprehensive approaches have established PLA2G4A as both a prognostic marker and a potential therapeutic target for AML with MLL rearrangement, offering new avenues for treatment of this aggressive malignancy .

How can researchers utilize PLA2G4A antibodies in neuropsychiatric disorder studies?

PLA2G4A antibodies enable sophisticated investigation of its role in neuropsychiatric disorders, particularly schizophrenia:

Copy number variation (CNV) correlation studies:

• Combine genetic CNV detection with protein expression analysis:

  • Identify PLA2G4A gene deletions using qPCR

  • Use ELISA with specific antibodies to measure peripheral blood cytosolic PLA2 (cPLA2) levels

  • Compare protein levels across different genotypes (normal, deletion, duplication)

Research has demonstrated a significant decrease in peripheral blood cPLA2 levels in schizophrenia patients with the PLA2G4A deletion genotype compared to those with normal and copy number duplicate genotypes .

Genotype-phenotype correlation:

• Use antibody-based protein quantification to:

  • Correlate PLA2G4A protein levels with clinical symptoms

  • Analyze gender-specific effects (e.g., poverty of thought in males, erotomanic delusion in females)

  • Study treatment response in relation to PLA2G4A status

This approach has revealed significant correlations between PLA2G4A deletion and specific clinical manifestations, suggesting functional consequences of altered PLA2G4A expression .

Methodological considerations for CNV-protein studies:

• Sample preparation for PLA2G4A protein quantification:

  • Collect peripheral blood samples in EDTA tubes

  • Isolate plasma or serum under standardized conditions

  • Store samples at -80°C to preserve protein integrity

• ELISA quantification protocol:

  • Use validated commercial kits with PLA2G4A antibodies

  • Include appropriate standards (3.13-200 U/mL detection range)

  • Perform technical replicates to ensure reproducibility

  • Calculate intra-assay (CV<10%) and inter-assay (CV<15%) precision

• Data analysis for CNV-protein correlation:

  • Normalize protein expression data

  • Use appropriate statistical tests for group comparisons

  • Consider confounding factors (age, medication, disease duration)

These methodologies have established that functional copy number deletion in the PLA2G4A gene is associated with the risk of schizophrenia and clinical phenotypes by reducing the expression of cPLA2, providing a potential biomarker of susceptibility to schizophrenia .

How can researchers resolve discrepancies between predicted and observed molecular weights when detecting PLA2G4A?

The consistent discrepancy between the calculated (85 kDa) and observed (100-110 kDa) molecular weight of PLA2G4A in Western blotting is a well-documented phenomenon that requires proper interpretation:

Causes of molecular weight discrepancy:

• Post-translational modifications:

  • Phosphorylation at multiple sites (particularly Ser505)

  • Other modifications affecting protein migration

• Protein structure and composition:

  • PLA2G4A contains domains that may affect SDS binding

  • Tertiary structure elements that resist complete denaturation

  • Protein hydrophobicity affecting SDS-PAGE migration

• Technical factors:

  • Gel percentage (7.5-10% gels are optimal for resolving PLA2G4A)

  • Running buffer composition

  • Voltage and running time

Experimental approaches to address discrepancies:

• Validation with positive controls:

  • Run HeLa or NIH-3T3 cell lysates as positive controls

  • These consistently show PLA2G4A at 100-110 kDa

• Phosphorylation analysis:

  • Run parallel samples with and without phosphatase treatment

  • Compare migration patterns to assess contribution of phosphorylation

• Antibody validation:

  • Use multiple antibodies targeting different epitopes

  • All should detect the same 100-110 kDa band

  • Consider antibodies against both total and phosphorylated forms

• Mass spectrometry confirmation:

  • Excise the 100-110 kDa band from the gel

  • Perform mass spectrometry analysis to confirm protein identity

  • Identify post-translational modifications

• Recombinant protein comparison:

  • Run purified recombinant PLA2G4A alongside cellular samples

  • Compare migration patterns

When interpreting Western blot results, researchers should consider the 100-110 kDa band as the correct representation of PLA2G4A, rather than expecting a band at the calculated 85 kDa position .

What are the most effective approaches for optimizing signal-to-noise ratio in PLA2G4A antibody applications?

Achieving optimal signal-to-noise ratio with PLA2G4A antibodies requires systematic optimization across multiple parameters:

For Western blotting:

• Blocking optimization:

  • Test different blocking agents (5% non-fat milk, 5% BSA, commercial blockers)

  • BSA may be superior for phospho-specific detection

  • Optimize blocking time (1-2 hours at room temperature or overnight at 4°C)

• Antibody dilution optimization:

  • Perform dilution series (e.g., 1:500, 1:1000, 1:2000, 1:4000)

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Optimize secondary antibody dilution (typically 1:5000-1:10000)

• Washing protocol enhancement:

  • Increase number of washes (minimum 3× for 5 minutes each)

  • Use fresh washing buffer

  • Consider addition of 0.1-0.2% Tween-20 to reduce background

• Detection system optimization:

  • Compare different enhanced chemiluminescence (ECL) reagents

  • For weak signals, consider using femto-sensitivity substrates

  • Optimize exposure times to prevent overexposure

For immunohistochemistry/immunofluorescence:

• Antigen retrieval optimization:

  • Compare TE buffer pH 9.0 with citrate buffer pH 6.0

  • Test different retrieval times (10-30 minutes)

  • Compare different retrieval methods (microwave, pressure cooker, water bath)

• Endogenous enzyme blocking:

  • For IHC: Block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • For tissues with high endogenous biotin: Use avidin-biotin blocking kit

• Background reduction strategies:

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization

  • Use serum from the same species as the secondary antibody

  • Consider using commercial background reducers

• Signal amplification options:

  • For weak signals, consider tyramide signal amplification

  • Use polymer-based detection systems for IHC

  • For IF, try higher antibody concentrations or longer incubation times

For ELISA applications:

• Antibody pair optimization:

  • Test different capture and detection antibody combinations

  • Optimize antibody concentrations with checkerboard titration

  • Consider using affinity-purified antibodies for reduced background

• Sample preparation refinement:

  • Proper sample dilution to fall within the standard curve

  • Centrifuge samples to remove particulates

  • Consider pre-absorption with irrelevant proteins

• Detection sensitivity enhancement:

  • Optimize substrate incubation time (15-20 minutes at 37°C)

  • Protect from light during chromogenic development

  • Use high-sensitivity substrates for low abundance targets

These systematic optimization approaches can significantly improve signal-to-noise ratio and produce more reliable, reproducible results with PLA2G4A antibodies across various applications.

What strategies can researchers employ to validate PLA2G4A antibody specificity in their experimental systems?

Comprehensive validation of PLA2G4A antibody specificity requires a multi-faceted approach to ensure reliable experimental outcomes:

Genetic manipulation-based validation:

• RNA interference approaches:

  • Design multiple siRNA/shRNA constructs targeting different regions of PLA2G4A mRNA

  • Transfect cells and confirm knockdown by qRT-PCR (80-90% reduction ideal)

  • Perform Western blotting using the antibody being validated

  • Quantify signal reduction (should correlate with mRNA reduction)

  • Include non-targeting scrambled controls

• CRISPR-Cas9 knockout validation:

  • Generate complete knockout cell lines using CRISPR-Cas9

  • Confirm gene editing by sequencing

  • Compare antibody signal between wild-type and knockout cells

  • Expected outcome: Complete absence of specific signal in knockout cells

  • Enhanced validation analysis could utilize the siRNA knockdown approach with box plot analysis of the results

Overexpression validation:

• Tagged construct expression:

  • Generate expression vectors with epitope-tagged PLA2G4A (FLAG, HA, GFP)

  • Transfect into cells with low endogenous expression

  • Perform parallel detection with:

    • Anti-tag antibody

    • PLA2G4A antibody being validated

  • Analyze colocalization and signal intensity correlation

  • Signal overlap between antibody staining and GFP-tagged protein can be evaluated for enhanced validation

Biochemical validation:

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide at different ratios

  • Include no-peptide control

  • Apply to identical samples

  • Expected outcome: Dose-dependent reduction in signal

  • Complete elimination of specific signal at high peptide concentrations

• Immunoprecipitation-mass spectrometry:

  • Perform immunoprecipitation with the PLA2G4A antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm PLA2G4A as the predominant protein identified

  • Analyze any co-precipitated proteins for relevance

Multi-antibody validation:

• Independent antibody comparison:

  • Use multiple antibodies targeting different epitopes of PLA2G4A:

    • N-terminal domain antibodies (AA 1-178)

    • Internal region antibodies

    • C-terminal domain antibodies

  • Compare staining patterns across applications (WB, IHC, IF)

  • Consistent results across independent antibodies strongly support specificity

  • For enhanced validation, compare staining of two or more independent antibodies directed towards independent epitopes on the protein