PLA2G4F Antibody, HRP conjugated

What is PLA2G4F and what role does it play in cellular functions?

PLA2G4F (Phospholipase A2, Group IVF) is a member of the cytosolic phospholipase A2 family that selectively hydrolyzes glycerophospholipids at the sn-2 position. This enzyme plays important roles in membrane lipid remodeling, inflammatory response regulation, and cellular signaling pathways. PLA2G4F shares structural similarities with other group IV PLA2 enzymes but has distinct substrate preferences and tissue distribution patterns. The protein is encoded by a gene located in humans with molecular weight of approximately 95 kDa . Understanding PLA2G4F's function is critical for researchers investigating lipid metabolism, inflammation processes, and related pathological conditions.

What are the key differences between PLA2G4F antibodies and other phospholipase A2 family antibodies?

PLA2G4F antibodies specifically target the Group IVF phospholipase, distinguishing it from other family members such as PLA2G4D. The primary differences lie in the epitope recognition regions and cross-reactivity profiles. While PLA2G4F antibodies typically target amino acid regions 74-299 of the human protein , other family member antibodies like PLA2G4D antibodies target different epitopes specific to their respective proteins . This specificity is crucial for experimental design, as using the correct antibody ensures accurate detection of your target protein without cross-reactivity with related family members. When selecting between phospholipase antibodies, researchers should carefully verify the target sequence and validation data to ensure appropriate specificity for their experimental system.

How is the specificity of PLA2G4F antibody, HRP conjugated verified?

The specificity of HRP-conjugated PLA2G4F antibodies is verified through multiple validation methods. These typically include:

• Western blot analysis comparing PLA2G4F-expressing and non-expressing cell lysates

• ELISA assays with recombinant PLA2G4F protein and related family members

• Immunohistochemistry with positive and negative control tissues

• Pre-adsorption controls with the immunizing peptide

The HRP-conjugated PLA2G4F antibody shows reactivity specifically with human samples as indicated in the product information . Validation data typically demonstrates a single band at approximately 95 kDa in Western blots of human samples, with minimal cross-reactivity to other phospholipase family members. Researchers should review these validation data before selecting an antibody for their specific application to ensure appropriate specificity.

What are the optimal conditions for using PLA2G4F antibody, HRP conjugated in ELISA applications?

For optimal use of HRP-conjugated PLA2G4F antibody in ELISA applications, the following protocol is recommended:

• Coat plates with capture antigen (recombinant PLA2G4F protein or sample) at 1-10 μg/mL in carbonate buffer (pH 9.6) overnight at 4°C

• Block with 3% BSA in PBS for 1 hour at room temperature

• Apply HRP-conjugated PLA2G4F antibody at a recommended dilution of 1:500 to 1:2000

• Incubate for 1-2 hours at room temperature with gentle shaking

• Wash extensively (4-5 times) with PBST (PBS + 0.05% Tween-20)

• Develop with TMB substrate and measure absorbance at 450 nm

Optimization may be required for specific experimental systems. A preliminary titration experiment using 2-fold serial dilutions from 1:100 to 1:3200 is recommended to determine the optimal antibody concentration for your specific assay conditions. The antibody shows optimal performance in the pH range of 7.2-7.6 and temperatures between 20-25°C.

How should PLA2G4F antibody, HRP conjugated be used for immunohistochemistry applications?

For immunohistochemistry (IHC) applications with HRP-conjugated PLA2G4F antibody, follow this methodological approach:

• Prepare paraffin-embedded or frozen tissue sections (4-6 μm thickness)

• For paraffin sections: Deparaffinize, rehydrate, and perform antigen retrieval (citrate buffer pH 6.0, 95°C for 20 minutes recommended)

• Block endogenous peroxidase with 3% H₂O₂ in methanol for 15 minutes

• Block non-specific binding with 5% normal serum in PBS for 1 hour

• Apply HRP-conjugated PLA2G4F antibody at a dilution of 1:20-1:200

• Incubate overnight at 4°C in a humidified chamber

• Wash with PBS (3 × 5 minutes)

• Develop with DAB substrate until optimal signal is achieved (typically 2-5 minutes)

• Counterstain with hematoxylin, dehydrate, and mount

For frozen sections, fixation with 4% paraformaldehyde for 10 minutes prior to blocking steps is recommended. Optimization of antibody dilution is crucial, as the optimal concentration may vary based on tissue type and fixation method. Positive control tissues known to express PLA2G4F should be included in each experiment.

What are the recommended storage conditions for maintaining PLA2G4F antibody, HRP conjugated activity?

To maintain optimal activity of HRP-conjugated PLA2G4F antibody, adhere to the following storage recommendations:

• Store unopened antibody at -20°C or -80°C as indicated in the product documentation

• After initial use, aliquot to avoid repeated freeze-thaw cycles

• Each aliquot should be sufficient for a single experiment

• For short-term storage (≤1 month), store at 4°C protected from light

• Avoid exposure to elevated temperatures, extreme pH conditions, and oxidizing agents

The antibody contains preservatives such as ProClin 300 (0.03%) in a buffer of 50% glycerol and 0.01M PBS at pH 7.4 . This formulation helps maintain stability, but activity will gradually decrease over time. HRP activity is typically stable for 6-12 months when stored properly. Perform a small-scale activity test if the antibody has been stored for an extended period before using in critical experiments.

How can non-specific background be minimized when using PLA2G4F antibody, HRP conjugated?

To minimize non-specific background when using HRP-conjugated PLA2G4F antibody, implement these methodological strategies:

• Optimize blocking conditions:

  • Increase blocking agent concentration (5-10% normal serum or BSA)

  • Extend blocking time to 1-2 hours at room temperature

  • Consider adding 0.1-0.3% Triton X-100 to the blocking solution for better penetration

• Optimize antibody dilution:

  • Perform titration experiments to determine the minimum effective concentration

  • Start with the recommended dilution range of 1:20-1:200 and adjust as needed

• Improve washing procedures:

  • Increase number of washes (5-6 times)

  • Extend washing duration (10 minutes per wash)

  • Use PBS with 0.05-0.1% Tween-20 for more effective washing

• Additional strategies:

  • Pre-adsorb the antibody with non-specific proteins

  • Include 1-5% of the host species serum in the antibody diluent

  • Treat samples with commercial background reducers if appropriate

A systematic approach to optimization is recommended, changing only one parameter at a time to identify the most effective conditions for your specific experimental system.

What are the potential causes of false negative results when using PLA2G4F antibody, HRP conjugated?

False negative results when using HRP-conjugated PLA2G4F antibody may stem from various methodological issues. The following table outlines common causes and recommended solutions:

If false negative results persist despite these adjustments, verify the presence of PLA2G4F in your samples using alternative detection methods, such as RT-PCR or mass spectrometry.

How can signal strength be enhanced when using PLA2G4F antibody, HRP conjugated for low abundance targets?

For enhancing signal strength when detecting low-abundance PLA2G4F with HRP-conjugated antibodies, employ these methodological approaches:

• Signal amplification systems:

  • Implement tyramide signal amplification (TSA), which can increase sensitivity by 10-100 fold

  • Use polymer-based detection systems that increase HRP molecules per binding event

  • Consider avidin-biotin amplification if using a biotinylated primary antibody

• Sample enrichment techniques:

  • Perform subcellular fractionation to concentrate target proteins

  • Use immunoprecipitation to enrich PLA2G4F before detection

  • Concentrate protein samples through TCA precipitation or similar methods

• Optimize detection conditions:

  • Extend substrate development time (monitor closely to prevent background increase)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Optimize exposure times for imaging (multiple exposures recommended)

• Reduce detection thresholds:

  • Use cooled CCD cameras for imaging with increased exposure time

  • Employ photomultiplier-based detection systems with increased sensitivity settings

  • Consider computational image enhancement with appropriate controls

When implementing signal enhancement strategies, always include appropriate controls to distinguish between specific signal enhancement and increased background.

How can PLA2G4F antibody, HRP conjugated be used in multiplex immunoassays with other phospholipase antibodies?

For multiplex immunoassay development using HRP-conjugated PLA2G4F antibody alongside other phospholipase antibodies, implement these methodological approaches:

• Sequential detection strategy:

  • Apply antibodies in order of sensitivity (least sensitive to most sensitive)

  • Use complete stripping between detection cycles (validate stripping efficiency)

  • Employ different chromogenic substrates for different HRP-conjugated antibodies

  • Document each detection step separately with appropriate imaging

• Spatial separation technique:

  • Use microarray or compartmentalized assay platforms

  • Apply different antibodies to distinct spatial regions

  • Develop a standardized normalization method across regions

• Spectral separation method:

  • Combine HRP-conjugated PLA2G4F antibody with antibodies conjugated to different enzymes (e.g., alkaline phosphatase)

  • Use substrates that produce spectrally distinct products

  • Implement multi-wavelength detection and spectral unmixing algorithms

For optimal results, validation experiments should demonstrate lack of cross-reactivity between antibodies and absence of signal interference. Cross-validation with single-antibody controls is essential to confirm that the multiplex assay provides equivalent results to individual assays.

What is the role of PLA2G4F in inflammatory responses, and how can PLA2G4F antibody, HRP conjugated contribute to this research?

PLA2G4F plays a significant role in inflammatory responses through regulation of arachidonic acid release and subsequent eicosanoid production. HRP-conjugated PLA2G4F antibody can contribute to this research through:

• Quantitative assessment of PLA2G4F expression:

  • Measure PLA2G4F protein levels in inflammatory versus normal tissues

  • Correlate expression with clinical parameters or disease severity

  • Monitor temporal changes in expression during inflammation progression

• Cellular localization studies:

  • Determine subcellular translocation during inflammatory activation

  • Analyze co-localization with inflammatory pathway components

  • Investigate cell type-specific expression in inflammatory microenvironments

• Functional association studies:

  • Correlate PLA2G4F localization with markers of inflammatory activation

  • Compare PLA2G4F expression with downstream inflammatory mediators

  • Evaluate potential associations with specific inflammatory pathways

Published research indicates that phospholipase A2 family members play critical roles in inflammatory diseases through lipid mediator generation. The HRP-conjugated PLA2G4F antibody enables sensitive detection of this protein in various inflammatory models, contributing to our understanding of its specific role compared to other family members.

How does the specificity of PLA2G4F antibody compare when detecting the protein in different experimental systems (cell lines vs. tissue samples)?

The specificity and sensitivity of HRP-conjugated PLA2G4F antibody varies across experimental systems due to several factors. The following table compares detection characteristics in different sample types:

The antibody shows reactivity with human samples as indicated in the product information . When transitioning between experimental systems, validation experiments should be performed to confirm specificity in each new system. Western blot analysis of both the new system and a validated positive control should be conducted in parallel to ensure consistent detection.

What are the latest research applications utilizing PLA2G4F antibody, HRP conjugated in studies of lipid metabolism disorders?

Recent research applications utilizing PLA2G4F antibodies in lipid metabolism disorders include:

• Metabolic disease investigations:

  • Quantification of PLA2G4F expression in adipose tissues from normal versus obese subjects

  • Correlation analysis between PLA2G4F levels and insulin resistance markers

  • Evaluation of altered PLA2G4F expression in non-alcoholic fatty liver disease

• Lipid signaling pathway analysis:

  • Investigation of PLA2G4F's role in generating specific lipid second messengers

  • Comparison with other phospholipase family members in specific lipid mobilization

  • Identification of unique substrate preferences through co-localization with lipid pools

• Therapeutic target evaluation:

  • Screening potential inhibitors of PLA2G4F activity

  • Monitoring PLA2G4F expression changes in response to treatments

  • Evaluating PLA2G4F as a biomarker for treatment response

HRP-conjugated antibodies facilitate sensitive detection in these applications through techniques like immunohistochemistry, ELISA, and Western blotting. The conjugation to HRP eliminates the need for secondary antibody incubation, reducing background and improving specific signal detection in complex lipid-rich samples.

What controls should be included when using PLA2G4F antibody, HRP conjugated for critical research applications?

For rigorous research applications utilizing HRP-conjugated PLA2G4F antibody, implement this comprehensive control strategy:

• Positive controls:

  • Cell lines or tissues with confirmed PLA2G4F expression

  • Recombinant PLA2G4F protein (full-length or the specific epitope region)

  • Previously validated samples with known signal patterns

• Negative controls:

  • PLA2G4F knockout or knockdown samples

  • Tissues known not to express PLA2G4F

  • Isotype control antibody (rabbit IgG, HRP-conjugated) at the same concentration

• Technical controls:

  • Antibody dilution series to confirm signal linearity

  • Peptide competition assay using the immunizing peptide (aa 74-299)

  • Primary antibody omission control to assess non-specific binding

• Validation controls:

  • Parallel detection with an alternative PLA2G4F antibody recognizing a different epitope

  • Correlation with mRNA expression (RT-PCR)

  • Blocking peptide controls to confirm specificity

Documentation of all control results is essential for publication-quality research. Quantitative assessment of signal-to-noise ratios across controls provides objective evaluation of assay performance and reliability.

How do different tissue fixation methods affect the performance of PLA2G4F antibody, HRP conjugated in immunohistochemistry?

Tissue fixation methodology significantly impacts the performance of HRP-conjugated PLA2G4F antibody in immunohistochemistry. The following table summarizes these effects:

For optimal results with the HRP-conjugated PLA2G4F antibody in immunohistochemistry applications, 4% paraformaldehyde fixation for 12-24 hours followed by paraffin embedding generally provides the best balance of tissue morphology preservation and epitope accessibility. For challenging samples, a comparative approach testing multiple fixation and retrieval methods may be necessary to determine optimal conditions.

How does the performance of PLA2G4F antibody, HRP conjugated compare with unconjugated primary antibodies in terms of sensitivity and specificity?

When comparing HRP-conjugated PLA2G4F antibody with unconjugated versions, several performance characteristics should be considered:

• Sensitivity comparison:

  • Direct HRP conjugation typically offers 1.5-2× improved sensitivity over two-step detection

  • Elimination of secondary antibody binding variability provides more consistent results

  • Signal amplification capability is generally reduced compared to multi-step detection systems

• Specificity analysis:

  • HRP conjugation may slightly reduce epitope recognition efficiency in some cases

  • Absence of secondary antibody eliminates potential cross-reactivity issues

  • Conjugation process may affect antibody binding kinetics

• Background considerations:

  • HRP-conjugated antibodies eliminate secondary antibody background

  • Direct conjugates typically show reduced non-specific tissue binding

  • Endogenous peroxidase blocking becomes more critical with direct HRP conjugates

What are the considerations for using PLA2G4F antibody, HRP conjugated in high-throughput screening applications?

For high-throughput screening applications using HRP-conjugated PLA2G4F antibody, implement these methodological considerations:

• Assay miniaturization strategies:

  • Optimize antibody concentration for reduced volume applications

  • Determine minimum required incubation times for adequate signal

  • Evaluate signal-to-noise ratio in miniaturized format

• Automation compatibility factors:

  • Assess antibody stability under robotic handling conditions

  • Validate performance consistency across batch processing

  • Implement quality control checkpoints at critical steps

• Data standardization methods:

  • Develop robust normalization procedures for plate-to-plate comparison

  • Include calibration standards on each plate

  • Establish signal threshold criteria for hit identification

• Practical implementation considerations:

  • Prepare single-use antibody aliquots to maintain consistency

  • Develop simplified washing protocols compatible with automation

  • Implement real-time monitoring of enzyme activity stability

HRP-conjugated antibodies offer particular advantages in high-throughput applications due to elimination of secondary antibody incubation steps, reducing handling time and potential variability. Recommended working dilutions for high-throughput microplate formats range from 1:500 to 1:2000 , with optimization suggested for specific assay conditions and detection systems.

How can PLA2G4F antibody, HRP conjugated be used to investigate the relationship between PLA2G4F expression and disease progression?

To investigate relationships between PLA2G4F expression and disease progression using HRP-conjugated antibodies, implement these methodological approaches:

• Longitudinal tissue analysis:

  • Evaluate PLA2G4F expression across disease stages using tissue microarrays

  • Quantify staining intensity using digital pathology and image analysis software

  • Correlate expression patterns with clinical progression metrics

• Mechanistic investigation techniques:

  • Compare PLA2G4F expression with markers of disease pathology

  • Analyze co-localization with relevant signaling pathway components

  • Evaluate relationship with lipid mediators using complementary mass spectrometry

• Clinical correlation strategies:

  • Develop standardized scoring systems for PLA2G4F immunoreactivity

  • Perform multivariate analysis with clinical parameters and outcomes

  • Validate findings across independent patient cohorts

The specificity of the HRP-conjugated PLA2G4F antibody for human samples makes it particularly valuable for translational research using patient-derived materials. When analyzing clinical samples, it is recommended to include normal adjacent tissue controls and establish baseline expression in healthy tissues for accurate comparative analysis.