PLA2G4F Antibody, Biotin conjugated

What is PLA2G4F and what is its biological function?

PLA2G4F (Phospholipase A2 Group IVF) is a calcium-dependent phospholipase A2 enzyme that selectively hydrolyzes glycerophospholipids in the sn-2 position. It demonstrates higher enzymatic activity toward phosphatidylethanolamine compared to phosphatidylcholine. This enzyme belongs to the cytosolic phospholipase A2 family and is involved in lipid metabolism pathways . The protein plays roles in signal transduction, inflammation, and membrane remodeling through the release of fatty acids from membrane phospholipids. Understanding its function is critical for research in metabolism, cancer biology, and inflammatory conditions .

What are the key characteristics of PLA2G4F Antibody, Biotin conjugated?

PLA2G4F Antibody, Biotin conjugated (Product Code: CSB-PA721207LD01HU) is a rabbit polyclonal IgG antibody generated against recombinant Human Cytosolic phospholipase A2 zeta protein (amino acids 74-299). The antibody is specifically reactive to human PLA2G4F protein (UniProt ID: Q68DD2) . The antibody has been affinity-purified and conjugated with biotin, making it suitable for detection systems utilizing streptavidin/avidin. It is supplied in liquid form containing a preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . Its biotin conjugation enables enhanced detection sensitivity in various immunoassay applications.

How should PLA2G4F Antibody, Biotin conjugated be stored to maintain optimal activity?

For optimal preservation of activity, PLA2G4F Antibody, Biotin conjugated should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation, loss of biotin conjugation efficiency, and ultimately decreased antibody performance . If frequent use is anticipated, it is recommended to prepare small working aliquots before freezing to minimize freeze-thaw cycles. The antibody formulation (50% glycerol with 0.01M PBS, pH 7.4) provides stability during frozen storage . For short-term storage (1-2 weeks), 4°C is acceptable, but extended storage at this temperature may lead to gradual loss of activity and should be avoided.

What are the primary applications for PLA2G4F Antibody, Biotin conjugated?

PLA2G4F Antibody, Biotin conjugated has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications as its primary use . While listed as tested specifically for ELISA, related PLA2G4F antibodies have demonstrated utility in Western Blot (WB) and Immunohistochemistry (IHC) applications . The biotin conjugation makes this antibody particularly valuable for streptavidin-based detection systems, which can provide signal amplification and enhanced sensitivity. This antibody is designed for research use only and should not be employed in diagnostic or therapeutic procedures . The versatility of biotin-conjugated antibodies allows researchers to use various detection methods including colorimetric, chemiluminescent, and fluorescent readouts depending on the streptavidin conjugate used.

How does PLA2G4F function differ from other phospholipase A2 family members?

PLA2G4F (cytosolic phospholipase A2 zeta) demonstrates distinct substrate specificity compared to other phospholipase A2 family members. Unlike PLA2G4A (cPLA2α) which preferentially cleaves arachidonic acid from membrane phospholipids, PLA2G4F shows higher enzyme activity for phosphatidylethanolamine than phosphatidylcholine . This substrate preference influences its biological role in lipid signaling networks. Additionally, while PLA2G4B (a related family member) exhibits calcium-dependent activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low lysophospholipase activity, its activity against phosphatidylcholine is significantly lower than other family members . These functional differences suggest specialized roles in maintaining membrane composition and generating specific lipid mediators during cellular responses to various stimuli.

What considerations should be made when designing experiments to study PLA2G4F interactions with other signaling proteins?

When designing experiments to study PLA2G4F interactions with other signaling proteins, researchers should consider:

• Calcium dependency: As a calcium-dependent enzyme, experimental buffers must contain appropriate calcium concentrations to maintain physiological activity .

• Membrane association: Consider using membrane fractionation techniques to isolate membrane-associated complexes where PLA2G4F likely functions.

• Co-immunoprecipitation approaches: When using biotin-conjugated PLA2G4F antibodies in co-IP experiments, streptavidin magnetic beads can be employed, but potential steric hindrance from the biotin conjugation should be evaluated .

• Crosslinking strategies: Consider reversible crosslinking approaches to capture transient protein-protein interactions.

• Signal transduction context: Design experiments that account for PLA2G4F's roles in cancer, metabolism, and signal transduction pathways .

• Controls: Include appropriate negative controls (isotype-matched biotin-conjugated IgG) and positive controls (known interaction partners) to validate experimental findings.

What are the current hypotheses regarding PLA2G4F's role in cancer biology?

Current research suggests multiple potential roles for PLA2G4F in cancer biology:

• Lipid metabolism alterations: Cancer cells often exhibit dysregulated lipid metabolism, and PLA2G4F may contribute to this metabolic reprogramming through its phospholipase activity, generating fatty acids and lysophospholipids that support cancer cell growth .

• Inflammatory microenvironment: The enzymatic products of PLA2G4F activity may contribute to the pro-inflammatory tumor microenvironment, promoting cancer progression through immune modulation.

• Signal transduction: PLA2G4F has been implicated in signal transduction pathways that may influence cell proliferation, survival, and metastatic potential in various cancer types .

• Membrane remodeling: Cancer cells require extensive membrane remodeling during proliferation and migration; PLA2G4F's phospholipase activity may facilitate these processes.

• Biomarker potential: Expression levels of PLA2G4F are being investigated as potential diagnostic or prognostic biomarkers in certain cancer types.

These hypotheses are actively being investigated using techniques like immunohistochemistry with anti-PLA2G4F antibodies to assess expression in tumor tissues compared to normal tissues .

What is the optimal protocol for using PLA2G4F Antibody, Biotin conjugated in ELISA assays?

Optimal protocol for PLA2G4F Antibody, Biotin conjugated in ELISA:

• Plate preparation: Coat high-binding ELISA plates with capture antigen (recombinant PLA2G4F or cellular lysates containing PLA2G4F) at 1-10 μg/mL in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Primary antibody incubation: Dilute PLA2G4F Antibody, Biotin conjugated (starting at 1:500 and titrating as needed) in blocking buffer and incubate for 1-2 hours at room temperature .

• Detection: Incubate with streptavidin-HRP (typically 1:2000 to 1:5000 dilution) for 30-60 minutes at room temperature.

• Substrate reaction: Add TMB substrate and allow color development (5-15 minutes), then stop with 2N H₂SO₄.

• Readout: Measure absorbance at 450 nm with reference at 620 nm.

Critical considerations:

• Determine optimal antibody dilution through titration experiments

• Include positive and negative controls

• Maintain consistent incubation times and temperatures

• Thorough washing between steps (3-5 times with PBS-T)

• The biotin conjugation eliminates the need for a secondary antibody, streamlining the protocol

How can PLA2G4F Antibody, Biotin conjugated be adapted for use in Western blotting?

While the PLA2G4F Antibody, Biotin conjugated is primarily tested for ELISA applications, it can be adapted for Western blotting with the following protocol adjustments:

• Sample preparation: Prepare cell/tissue lysates with protease inhibitors. Consider phosphatase inhibitors if phosphorylation status is relevant.

• Gel electrophoresis: Separate proteins using SDS-PAGE. The expected molecular weight of PLA2G4F is approximately 95 kDa .

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard methods.

• Blocking: Block membrane with 3-5% BSA or non-fat dry milk in TBS-T for 1 hour at room temperature.

• Primary antibody: Incubate membrane with PLA2G4F Antibody, Biotin conjugated (1:500 to 1:2000 dilution, optimized through titration) in blocking buffer overnight at 4°C.

• Detection: Incubate with streptavidin-HRP (1:2000 to 1:5000) for 1 hour at room temperature.

• Visualization: Develop using ECL substrate and image using appropriate detection system.

Optimization considerations:

• Test different blocking agents (BSA vs. milk) to reduce background

• Include positive control (cell lines known to express PLA2G4F)

• Consider non-reducing conditions if epitope recognition is affected by disulfide reduction

• The biotin-streptavidin interaction provides signal amplification, which may be beneficial for detecting low-abundance targets

What controls should be included when using PLA2G4F Antibody, Biotin conjugated in immunohistochemistry?

When adapting PLA2G4F Antibody, Biotin conjugated for immunohistochemistry, the following controls are essential:

• Positive tissue control: Include tissues known to express PLA2G4F (based on literature or previous validation). This confirms the ability of the antibody to detect the target under the specific experimental conditions.

• Negative tissue control: Include tissues known not to express PLA2G4F to confirm specificity.

• Isotype control: Use a biotin-conjugated rabbit IgG isotype control at the same concentration as the PLA2G4F antibody to identify non-specific binding .

• Absorption control: Pre-incubate the PLA2G4F Antibody with excess recombinant PLA2G4F protein (the immunogen used to generate the antibody) before IHC staining to confirm specificity.

• Endogenous biotin blocking: Use avidin/biotin blocking kit to minimize background from endogenous biotin, particularly important in tissues like liver, kidney, and brain.

• Endogenous peroxidase quenching: If using HRP-based detection, block endogenous peroxidase activity with hydrogen peroxide treatment before antibody incubation.

• No primary antibody control: Omit the primary antibody but include all other steps to assess background from the detection system.

These controls help distinguish between specific PLA2G4F staining and various sources of background or non-specific signals .

What are common troubleshooting approaches for weak or absent signal when using PLA2G4F Antibody, Biotin conjugated?

How should researchers approach quantitative analysis of PLA2G4F expression across different experimental conditions?

Quantitative analysis of PLA2G4F expression requires systematic approaches tailored to the experimental technique:

• For Western blot quantification:

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Employ densitometric analysis software

  • Generate standard curves using recombinant PLA2G4F protein

  • Normalize PLA2G4F signal to total protein (using stain-free technology or total protein stains)

  • Run biological replicates (minimum n=3) for statistical validation

• For ELISA quantification:

  • Generate standard curves using recombinant PLA2G4F protein at known concentrations

  • Ensure all samples fall within the linear range of detection

  • Run all samples in technical duplicates or triplicates

  • Calculate intra- and inter-assay coefficients of variation

  • Use four-parameter logistic regression for curve fitting

• For immunohistochemical quantification:

  • Employ digital image analysis software for unbiased quantification

  • Use standardized scoring systems (H-score, Allred score) for semi-quantitative analysis

  • Conduct blind scoring by multiple independent observers

  • Correlate IHC results with other quantitative methods (Western blot, qPCR)

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological versus technical variation

  • Report both absolute and relative changes in expression levels

What are potential sources of data inconsistency when comparing PLA2G4F expression across different experimental platforms?

When comparing PLA2G4F expression across different experimental platforms, researchers should consider these potential sources of inconsistency:

• Antibody epitope differences:

  • Different antibodies may recognize distinct epitopes within PLA2G4F, resulting in variable detection efficiency

  • The biotin conjugation might affect epitope recognition in certain contexts

• Post-translational modifications:

  • Phosphorylation or other modifications may affect antibody binding differently across techniques

  • Sample preparation methods might preserve or disrupt these modifications

• Isoform specificity:

  • PLA2G4F may exist in multiple isoforms, and different detection methods may have varying sensitivities to specific isoforms

• Sample preparation variations:

  • Protein denaturation in Western blotting versus native conformation in ELISA

  • Fixation methods in IHC may alter epitope accessibility

  • Cell lysis buffers may extract PLA2G4F with different efficiencies

• Detection sensitivity thresholds:

  • ELISA typically offers higher sensitivity than Western blot

  • Signal amplification from biotin-streptavidin interaction varies across platforms

• Quantification methodologies:

  • Densitometry for Western blot versus absorbance measurements in ELISA

  • Subjective scoring versus digital analysis in IHC

• Reference standards:

  • Inconsistent use of housekeeping genes or normalization methods

  • Lack of absolute quantification standards across platforms

How can PLA2G4F Antibody, Biotin conjugated be utilized in multiplex immunoassays with other biomarkers?

PLA2G4F Antibody, Biotin conjugated can be strategically incorporated into multiplex immunoassays through these approaches:

• Multiplexed immunofluorescence:

  • Combine with differently labeled antibodies against related signaling proteins

  • Use streptavidin conjugated to spectrally distinct fluorophores (Alexa Fluor 488, 555, 647)

  • Implement sequential staining protocols to avoid cross-reactivity

  • Employ multispectral imaging systems for signal separation

• Bead-based multiplex assays:

  • Couple capture antibodies for different targets to spectrally distinct beads

  • Use PLA2G4F Antibody, Biotin conjugated as one detection antibody in the panel

  • Develop with streptavidin-PE or streptavidin-APC

  • Analyze using flow cytometry or dedicated multiplex readers

• Sequential multiplex chromogenic IHC:

  • Use orthogonal detection systems (HRP, AP) with different chromogens

  • Incorporate the biotin-conjugated antibody in one detection round

  • Consider tyramide signal amplification for enhanced sensitivity

  • Apply spectral unmixing algorithms for quantitative analysis

• Practical considerations:

  • Validate antibody performance in singleplex before multiplexing

  • Test for cross-reactivity between antibodies in the panel

  • Optimize signal-to-noise ratios for each biomarker

  • Include appropriate controls for each target protein

  • Consider potential steric hindrance in simultaneous detection protocols

What methodological approaches can be used to study PLA2G4F enzyme activity in conjunction with expression analysis?

Comprehensive analysis of PLA2G4F should combine expression and activity measurements using these methodological approaches:

This integrated approach helps distinguish between changes in enzyme expression versus activity regulation through post-translational modifications.

How can computational approaches complement experimental data when studying PLA2G4F structure-function relationships?

Computational approaches provide valuable insights into PLA2G4F structure-function relationships that complement experimental findings:

• Homology modeling and structural prediction:

  • Generate 3D structural models of PLA2G4F based on homologous phospholipases

  • Predict the catalytic site and substrate binding pocket architecture

  • Identify conserved domains shared with other phospholipase A2 family members

  • Use AlphaFold or RoseTTAFold for accurate structure prediction

• Molecular dynamics simulations:

  • Investigate conformational changes upon calcium binding

  • Simulate interactions with membrane phospholipids

  • Analyze the dynamic behavior of the catalytic site

  • Predict effects of experimental mutations on enzyme function

• Protein-protein interaction networks:

  • Predict functional protein associations using STRING or similar databases

  • Identify potential binding partners in signaling cascades

  • Develop testable hypotheses about regulatory interactions

  • Construct pathway models integrating PLA2G4F

• Structure-based drug design:

  • Virtual screening for potential PLA2G4F inhibitors

  • Molecular docking to predict binding modes

  • Pharmacophore modeling to identify key interaction features

  • Guide rational design of selective inhibitors

• Integration with experimental data:

  • Use computational predictions to guide site-directed mutagenesis experiments

  • Validate structural models with limited proteolysis and mass spectrometry

  • Compare substrate specificity predictions with experimental enzyme kinetics

  • Correlate predicted structural features with immunological epitopes recognized by the biotin-conjugated antibody

What emerging techniques might enhance the utility of PLA2G4F Antibody, Biotin conjugated in single-cell analysis?

Emerging technologies are expanding the applications of biotin-conjugated antibodies like PLA2G4F in single-cell analysis:

• Mass cytometry (CyTOF) integration:

  • Conjugate streptavidin with rare earth metal isotopes

  • Combine with other metal-tagged antibodies for high-parameter analysis

  • Achieve 40+ parameter analysis without fluorescence spillover concerns

  • Enable deep phenotyping of heterogeneous cell populations

• Spatial transcriptomics combinations:

  • Use PLA2G4F antibody detection alongside in situ transcriptomics

  • Correlate protein expression with mRNA levels at single-cell resolution

  • Implement sequential immunofluorescence and RNA-FISH protocols

  • Leverage biotin-streptavidin amplification for enhanced sensitivity

• Microfluidic applications:

  • Incorporate into droplet-based single-cell protein assays

  • Develop microfluidic antibody capture for secreted phospholipase products

  • Create integrated platforms for simultaneous analysis of multiple cellular parameters

  • Combine with single-cell Western blotting technologies

• Advanced microscopy integration:

  • Apply super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Implement light-sheet microscopy for rapid 3D imaging with minimal phototoxicity

  • Utilize expansion microscopy to physically magnify subcellular structures

  • Combine with live-cell imaging using photoconvertible streptavidin conjugates

• AI-enhanced image analysis:

  • Deploy deep learning algorithms for automated single-cell segmentation

  • Develop machine learning models for phenotype classification

  • Implement advanced spatial statistics for quantifying co-localization patterns

  • Create predictive models linking PLA2G4F localization to cellular functions

What are the key research questions regarding PLA2G4F's role in disease pathogenesis that remain to be addressed?

Despite advances in understanding PLA2G4F, several critical research questions remain unresolved:

• Cancer biology:

  • How does PLA2G4F expression correlate with specific cancer subtypes and stages?

  • What is the prognostic significance of PLA2G4F expression in various malignancies?

  • Does PLA2G4F contribute to therapy resistance mechanisms?

  • How does PLA2G4F activity influence the tumor microenvironment?

• Inflammatory disorders:

  • What is PLA2G4F's specific contribution to eicosanoid production compared to other PLA2 isoforms?

  • Does selective inhibition of PLA2G4F offer therapeutic advantages in inflammatory diseases?

  • How is PLA2G4F expression regulated during acute versus chronic inflammation?

  • What are the tissue-specific roles of PLA2G4F in inflammatory pathology?

• Metabolic regulation:

  • How does PLA2G4F contribute to lipid metabolism disorders?

  • What is its role in cellular energy homeostasis?

  • Does PLA2G4F activity change in obesity or diabetes?

  • How does nutritional status affect PLA2G4F expression and function?

• Signal transduction mechanisms:

  • What are the upstream regulators of PLA2G4F activity?

  • How is PLA2G4F integrated into established signaling networks?

  • What post-translational modifications regulate PLA2G4F activity?

  • Do specific lipid products of PLA2G4F have signaling functions distinct from other PLA2 enzymes?

• Therapeutic targeting:

  • Can selective PLA2G4F inhibitors be developed as potential therapeutics?

  • What are potential off-target effects of PLA2G4F modulation?

  • Could PLA2G4F serve as a biomarker for disease diagnosis or treatment response?

  • What combination therapies might enhance the efficacy of PLA2G4F targeting?

How might technological advances in antibody engineering enhance future generations of PLA2G4F detection tools?

Emerging antibody engineering technologies promise to create enhanced PLA2G4F detection tools:

• Recombinant antibody formats:

  • Single-chain variable fragments (scFv) for improved tissue penetration

  • Bispecific antibodies targeting PLA2G4F and related pathway components

  • Nanobodies offering smaller size and unique epitope recognition

  • Intrabodies designed for tracking intracellular PLA2G4F

• Site-specific conjugation strategies:

  • Enzymatic biotinylation at defined sites to preserve antigen binding

  • Click chemistry approaches for controlled biotin-to-antibody ratios

  • Sortase-mediated conjugation for oriented biotin positioning

  • Incorporation of unnatural amino acids for precise modification

• Sensitivity enhancements:

  • DNA-barcoded antibodies for ultrasensitive digital detection

  • Proximity ligation assays to detect PLA2G4F interactions

  • Photoswitchable fluorescent conjugates for super-resolution imaging

  • Electrochemiluminescent detection systems with femtomolar sensitivity

• Multiplexing capabilities:

  • Spectral barcoding of multiple epitopes on PLA2G4F

  • Mass-tagged antibodies for high-parameter analysis

  • DNA-encoded antibody libraries for massively parallel epitope mapping

  • Cyclic immunofluorescence for sequential detection of dozens of targets

• Functional reporting:

  • Activity-sensing antibodies that detect conformational changes

  • Split-reporter systems activated upon target binding

  • Antibody-enzyme fusions for localized signal amplification

  • FRET-based biosensors reporting on PLA2G4F-substrate interactions

These technological advances will enable not just detection of PLA2G4F but provide deeper insights into its functional states, interaction partners, and roles in various cellular contexts .