PLA2G4B Antibody, FITC conjugated

What is PLA2G4B and why is it important in biochemical research?

Phospholipase A2, Group IVB (Cytosolic) (PLA2G4B) is a member of the cytosolic phospholipase A2 family that plays crucial roles in phospholipid metabolism and signal transduction pathways. This enzyme catalyzes the hydrolysis of membrane phospholipids to release arachidonic acid, which serves as a precursor for eicosanoid production involved in inflammatory responses. Research into PLA2G4B contributes to understanding cellular signaling mechanisms, inflammatory processes, and potential therapeutic targets. Antibodies against PLA2G4B, such as the FITC-conjugated variant, enable researchers to visualize and quantify this protein in various experimental contexts, facilitating investigations into its expression patterns, subcellular localization, and functional interactions .

What are the key specifications of the PLA2G4B Antibody, FITC conjugated?

The PLA2G4B Antibody, FITC conjugated (e.g., ABIN7149406) is a polyclonal antibody raised in rabbits that specifically targets amino acids 514-781 of the human Phospholipase A2, Group IVB protein. This antibody has been purified using Protein G purification technology, achieving >95% purity. The antibody is of IgG isotype and has been immunogen-affinity purified using recombinant Human Cytosolic phospholipase A2 beta protein (514-781AA) as the immunogen. The antibody has been validated for reactivity with human samples, making it suitable for experiments involving human cell lines or tissues .

What applications is the PLA2G4B Antibody, FITC conjugated suitable for?

While the manufacturer indicates "Please inquire" regarding specific applications for this particular FITC-conjugated antibody, related PLA2G4B antibodies are commonly used in several techniques. Based on the properties of FITC-conjugated antibodies and the characteristics of this specific antibody, it would likely be suitable for immunofluorescence (IF), flow cytometry (FACS), and potentially ELISA applications. The antibody's specificity for amino acids 514-781 makes it particularly valuable for experiments requiring detection of specific domains of the PLA2G4B protein. Researchers should verify application-specific protocols before proceeding with experiments, as optimization may be required for different experimental systems .

How should researchers optimize immunofluorescence protocols when using PLA2G4B Antibody, FITC conjugated?

Optimization of immunofluorescence protocols when using FITC-conjugated PLA2G4B antibody requires systematic evaluation of several parameters:

• Fixation method: Different fixation approaches (e.g., paraformaldehyde, methanol, acetone) can significantly affect epitope accessibility. Test multiple fixation methods to determine which best preserves the target epitope (AA 514-781).

• Permeabilization: Since PLA2G4B is cytosolic, appropriate permeabilization is critical. A recommended approach is to test a concentration series of permeabilization agents (e.g., 0.1-0.5% Triton X-100 or 0.05-0.2% Saponin) to determine optimal conditions.

• Blocking conditions: To minimize non-specific binding, implement a robust blocking step using 5-10% normal serum from a species different from the antibody host (non-rabbit), or 3-5% BSA in PBS-T.

• Antibody dilution: Conduct a titration series (e.g., 1:50, 1:100, 1:200, 1:500) to identify the dilution that provides optimal signal-to-noise ratio.

• Incubation conditions: Compare varying incubation times (1h, 2h, overnight) and temperatures (room temperature vs. 4°C) to determine optimal binding conditions.

• Counterstaining: When co-staining with other markers, ensure FITC signal (excitation ~495nm, emission ~519nm) is compatible with other fluorophores to prevent spectral overlap .

What validation approaches should be implemented to confirm antibody specificity?

Comprehensive validation of the FITC-conjugated PLA2G4B antibody specificity requires multiple complementary approaches:

• Positive and negative controls: Include known positive samples (tissues/cells with confirmed PLA2G4B expression) and negative controls (tissues/cells with confirmed absence of PLA2G4B or those treated with PLA2G4B-targeting siRNA).

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptide corresponding to the immunogen (amino acids 514-781) before application to samples. Specific signal should be significantly reduced or eliminated.

• Knockout validation: If available, test the antibody on PLA2G4B knockout cells or tissues; specific signal should be absent.

• Western blot correlation: When possible, correlate immunofluorescence results with Western blot analysis using unconjugated PLA2G4B antibodies (such as ABIN7117477) to confirm detection of a protein of the expected molecular weight.

• Cross-reactivity assessment: Test the antibody on samples from species other than human to evaluate potential cross-reactivity, especially if sequence conservation exists in the 514-781 amino acid region across species .

How can researchers troubleshoot weak or non-specific signals?

When encountering signal issues with FITC-conjugated PLA2G4B antibody, implement the following methodological troubleshooting approaches:

For weak signal:

• Increase antibody concentration (reduce dilution) while monitoring background levels

• Extend incubation time (e.g., overnight at 4°C instead of 1 hour at room temperature)

• Optimize antigen retrieval if working with fixed tissues

• Switch to a more sensitive detection system if using for applications beyond direct fluorescence

• Ensure samples have been appropriately stored to preserve protein integrity

For non-specific signal:

• Increase blocking stringency (longer time, higher concentration of blocking agent)

• Reduce antibody concentration (increase dilution)

• Add 0.1-0.3% Tween-20 to washing buffers to reduce hydrophobic interactions

• Perform additional washing steps (increase number and duration)

• Use a different fixation method that may better preserve epitope specificity

• If nuclear staining appears, add DNase treatment to eliminate potential DNA binding .

What controls are essential when using PLA2G4B Antibody, FITC conjugated?

A methodologically sound experimental design must include the following controls:

• Primary antibody controls:

  • Positive tissue/cell control (confirmed PLA2G4B expression)

  • Negative tissue/cell control (confirmed absence of PLA2G4B)

  • Concentration-matched isotype control (FITC-conjugated rabbit IgG)

• Technical controls:

  • Autofluorescence control (untreated sample to assess natural fluorescence)

  • Single-color controls (when performing multicolor experiments)

  • No-primary antibody control (to assess non-specific binding)

• Quantitative controls:

  • Standardized fluorescent beads (for flow cytometry applications)

  • Reference standards with known expression levels

  • Internal expression control (consistently expressed protein across samples)

These controls enable proper interpretation of results and facilitate troubleshooting of technical issues that may arise during experimentation .

How does the binding to amino acids 514-781 affect experimental design and result interpretation?

The binding specificity to amino acids 514-781 of PLA2G4B has several important implications for experimental design:

• Isoform detection: This antibody targets a specific region of PLA2G4B, which may not be present in all splice variants or heavily post-translationally modified forms of the protein. Researchers should verify whether their experimental system expresses variants containing this region.

• Epitope accessibility: The 514-781 region may be differentially accessible depending on protein conformation, interaction partners, or cellular conditions. This could lead to variable detection efficiency across different experimental contexts.

• Functional domain consideration: Researchers should determine whether this region encompasses or is adjacent to known functional domains, active sites, or protein-protein interaction regions of PLA2G4B, as antibody binding might interfere with these functions in live-cell applications.

• Experiment-specific limitations: In protocols involving protein denaturation (like Western blotting), linear epitopes within this region may be more accessible than in native-state applications like immunoprecipitation.

• Cross-reactivity assessment: Sequence alignment of this region (AA 514-781) with other phospholipase family members or unrelated proteins should be performed to anticipate potential cross-reactivity .

What dilution strategies should be employed for different experimental applications?

For all applications, researchers should perform a titration series to determine the optimal antibody concentration for their specific experimental system. The optimal dilution provides the highest specific signal while maintaining minimal background. When switching between different sample types (e.g., cell lines to tissues), re-optimization is recommended .

How can researchers quantitatively analyze data generated using PLA2G4B Antibody, FITC conjugated?

Quantitative analysis of data generated with FITC-conjugated PLA2G4B antibody requires systematic methodological approaches tailored to specific applications:

For microscopy-based quantification:

• Use standardized image acquisition parameters (exposure time, gain, laser power)

• Implement background subtraction using areas without specific staining

• Apply appropriate thresholding methods to distinguish positive from negative signals

• Quantify parameters such as:

  • Mean fluorescence intensity (within defined regions)

  • Percentage of positive cells

  • Subcellular distribution profiles (nuclear/cytoplasmic ratios)

  • Colocalization coefficients with other markers (Pearson's, Mander's)

For flow cytometry quantification:

• Use standardized fluorescent beads for day-to-day calibration

• Apply consistent gating strategies informed by negative controls

• Report data as:

  • Percentage of positive cells above threshold

  • Mean/median fluorescence intensity (MFI)

  • Molecules of equivalent soluble fluorochrome (MESF)

In all cases, statistical analysis should account for experimental variability and incorporate appropriate statistical tests based on data distribution and experimental design .

What approaches should be used to interpret different staining patterns?

Interpretation of staining patterns obtained with FITC-conjugated PLA2G4B antibody requires careful analysis of subcellular distribution and signal characteristics:

• Cytoplasmic diffuse pattern: Consistent with PLA2G4B's primarily cytosolic localization. Evaluate whether the pattern is uniform or shows regional intensity differences.

• Membrane-associated pattern: May indicate PLA2G4B recruitment to membranes during signaling events. Assess colocalization with membrane markers to confirm.

• Perinuclear accumulation: Could suggest association with endoplasmic reticulum or Golgi apparatus. Consider counterstaining with organelle markers.

• Punctate cytoplasmic pattern: May represent association with vesicular structures or specific signaling complexes.

• Nuclear staining: Could indicate either non-specific binding or previously unreported nuclear translocation. Requires careful validation using controls.

Pattern analysis should consider:

• Cell type-specific variations

• Cell cycle stage effects

• Activation state of signaling pathways

• Consistency across multiple samples

• Correlation with biochemical fractionation data when available .

How can researchers integrate PLA2G4B Antibody data with other phospholipase research approaches?

Integration of PLA2G4B antibody data with complementary research approaches enhances the validity and depth of phospholipase research:

• Correlative enzyme activity assays: Combine immunofluorescence detection of PLA2G4B expression with biochemical measurements of phospholipase A2 activity to establish expression-function relationships.

• Multi-omics integration: Correlate protein localization/expression data with:

  • Transcriptomic data on PLA2G4B expression

  • Lipidomic profiles of arachidonic acid and related metabolites

  • Phosphoproteomic data on PLA2G4B phosphorylation state

• Pathway analysis: Analyze PLA2G4B in the context of complete signaling pathways by combining with:

  • Phosphorylation-state specific antibodies against known regulators

  • Downstream eicosanoid production measurements

  • Calcium signaling dynamics

• Structural biology correlation: Interpret antibody binding data in the context of available structural information about PLA2G4B domains.

• Pharmacological manipulation: Combine antibody-based detection with specific inhibitors or activators of PLA2G4B to establish causal relationships in observed phenomena.

This integrated approach provides a more comprehensive understanding of PLA2G4B's role in cellular processes and disease mechanisms .

What emerging applications might benefit from PLA2G4B Antibody, FITC conjugated?

Several innovative research applications could potentially leverage the capabilities of FITC-conjugated PLA2G4B antibodies:

• Live-cell phospholipase dynamics: Modified protocols using reduced antibody concentrations may enable real-time visualization of PLA2G4B translocation during cellular activation, particularly in response to calcium signaling or inflammatory stimuli.

• Super-resolution microscopy: The FITC conjugate can be utilized in techniques such as STED, STORM, or PALM to achieve nanoscale resolution of PLA2G4B localization within specialized membrane compartments.

• Microfluidic single-cell analysis: Combining flow cytometry with microfluidic systems could enable correlation of PLA2G4B expression levels with functional outputs at the single-cell level.

• Tissue-based diagnostics: Development of standardized immunofluorescence protocols could potentially enable assessment of PLA2G4B expression patterns in clinical biopsies from inflammatory or neurodegenerative conditions.

• Biosensor development: The antibody could be adapted for use in FRET-based biosensors to detect conformational changes in PLA2G4B upon activation.

These emerging applications demonstrate the continuing value of well-characterized antibody reagents in advancing our understanding of phospholipase biology and its clinical implications .

How should researchers document and report PLA2G4B Antibody usage in publications?

To ensure experimental reproducibility and transparency, researchers should document the following details when reporting experiments using FITC-conjugated PLA2G4B antibody:

• Complete antibody identification information:

  • Catalog number (e.g., ABIN7149406)

  • Manufacturer/supplier

  • Clone/lot number

  • Host species and clonality (rabbit polyclonal)

  • Specific epitope targeted (AA 514-781)

  • Conjugation chemistry (FITC)

• Detailed methodological parameters:

  • Working dilution used for each application

  • Incubation conditions (time, temperature)

  • Buffer compositions

  • Sample preparation methods (fixation, permeabilization)

  • Image acquisition settings (exposure, gain, objective)

  • Flow cytometer settings and calibration methods

• Validation evidence:

  • Controls employed (positive, negative, isotype)

  • Supporting experiments confirming specificity

  • Reference to previous validation if applicable

• Data analysis approaches:

  • Software and algorithms used

  • Quantification methods

  • Statistical approaches

This comprehensive documentation ensures that other researchers can critically evaluate and potentially reproduce the experiments, advancing collective scientific understanding of PLA2G4B biology .

What complementary research tools should be considered alongside PLA2G4B antibody studies?

A comprehensive research approach to PLA2G4B should incorporate multiple complementary methodologies: