PLA2G4B Antibody, Biotin conjugated

What is PLA2G4B and why is it a target for research?

PLA2G4B (Phospholipase A2, Group IVB Cytosolic) is a calcium-dependent phospholipase that selectively hydrolyzes glycerophospholipids in the sn-2 position with a preference for arachidonoyl phospholipids. It has gained research interest due to its involvement in lipid metabolism and inflammation pathways. PLA2G4B has been implicated in psoriasis progression through activation of IL-17 production via the CD1b lipid antigen pathway, making it a potential therapeutic target for inflammatory skin conditions . The protein has a much weaker enzymatic activity compared to PLA2G4A, with different isoforms showing varying substrate specificities .

What is the relationship between JMJD7 and PLA2G4B genes?

PLA2G4B transcribes naturally-occurring mRNAs that are co-transcribed products of the neighboring JMJD7 and PLA2G4B genes. Incompletely processed read-through transcripts from these two loci are abundantly expressed in most tissues, creating fusion proteins that share amino acid sequences with each individual gene product. These transcripts include a partial JmjC domain and downstream C2 and phospholipase A2 domains, though the full function of this readthrough product has not been definitively determined .

What are the key specifications of commercially available PLA2G4B Antibody, Biotin conjugated?

The biotin-conjugated PLA2G4B antibody is typically a rabbit-derived polyclonal antibody targeting amino acids 396-424 or 514-781 from human PLA2G4B. It is affinity purified (>95% purity using Protein G) and supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), with 0.03% Proclin 300 as a preservative . The antibody is reactive with human samples and applicable for several detection methods including ELISA, Western Blotting, and in some formulations, Flow Cytometry .

What are the validated applications for PLA2G4B Antibody, Biotin conjugated?

Based on manufacturer validations, the biotin-conjugated PLA2G4B antibody has been tested and approved for:

• ELISA: As the primary detection method across all manufacturers

• Western Blotting (WB): Recommended dilution typically 1-5 μg/mL

• Flow Cytometry (FACS): Validated in certain formulations

For immunohistochemistry and immunofluorescence applications, verification is recommended as these applications vary between different epitope-targeting antibodies .

How should I optimize Western blot protocols when using PLA2G4B Antibody, Biotin conjugated?

For Western blot optimization with biotin-conjugated PLA2G4B antibody:

• Sample preparation: Use fresh samples with protease inhibitors to prevent degradation

• Protein loading: 20-40 μg of total protein is typically sufficient

• Blocking: Use 5% BSA in TBST rather than milk (which contains endogenous biotin)

• Primary antibody dilution: Start with 1-2 μg/mL and adjust based on signal strength

• Detection: Use streptavidin-HRP conjugate (1:5000-1:10000) as secondary detection reagent

• Expected molecular weight: ~53 kDa for the main PLA2G4B protein band

• Controls: Include a positive control tissue with known PLA2G4B expression

Note that optimization might be necessary as the antibody targets a readthrough protein that may show tissue-specific expression patterns .

What are the methodological considerations for ELISA using this antibody?

When developing an ELISA method with biotin-conjugated PLA2G4B antibody:

• Coating concentration: For capture antibody approach, use 1-10 μg/mL of non-biotinylated anti-PLA2G4B

• Blocking agent: Use biotin-free blocking solution (e.g., BSA or casein-based)

• Sample dilution: Prepare a dilution series to determine optimal concentration

• Detection: Utilize streptavidin-HRP system (avoid avidin due to higher background)

• Substrate: TMB substrate provides sensitive detection with biotinylated antibodies

• Standard curve: Recombinant PLA2G4B protein (514-781AA region) can serve as a calibrator

• Cross-reactivity: The antibody is human-specific; non-human samples may not yield reliable results

How can I address high background issues when using biotinylated PLA2G4B antibody?

High background is a common challenge with biotinylated antibodies. Consider these solutions:

• Pre-block endogenous biotin using a biotin blocking kit (especially important for tissues rich in endogenous biotin like liver, kidney, and brain)

• Increase washing steps (5-6 washes of 5 minutes each)

• Decrease antibody concentration (try a 1:2 or 1:5 dilution from your current concentration)

• Use streptavidin-based detection systems rather than avidin (less non-specific binding)

• Include 0.1-0.5% Tween-20 in wash buffers to reduce hydrophobic interactions

• For immunohistochemistry, perform antigen retrieval at pH 6.0 rather than pH 9.0

• Prepare fresh buffers before each experiment to prevent contamination

What cross-reactivity testing has been performed with this antibody?

The biotin-conjugated PLA2G4B antibody has been primarily validated for human samples. Cross-reactivity testing with mouse, rat, and other mammalian species varies by manufacturer and epitope target. Antibodies targeting the 396-424 amino acid region typically show human specificity, while antibodies targeting other regions may cross-react with mouse and rat samples . Researchers should validate cross-reactivity when working with non-human samples, especially since PLA2G4B contains readthrough transcripts that may have species-specific variations .

How should I store and handle PLA2G4B Antibody, Biotin conjugated to maintain activity?

To preserve antibody activity:

• Storage temperature: Maintain at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles (prepare small aliquots upon receipt)

• Working dilutions should be prepared fresh and used within 24 hours

• Protect from light (biotin conjugates can be light-sensitive)

• Shipping conditions: The antibody is typically shipped on dry ice

• Buffer components: The antibody is supplied in 50% glycerol with preservatives, making it stable at -20°C for at least 12 months from date of receipt

• Do not store diluted antibody for future use

How can PLA2G4B Antibody, Biotin conjugated be used to study its role in psoriasis?

Building on findings implicating PLA2G4B in psoriasis , researchers can:

• Employ the antibody for immunohistochemical analysis of psoriatic skin lesions versus healthy controls

• Use flow cytometry to quantify PLA2G4B expression in isolated skin-infiltrating immune cells

• Develop co-immunoprecipitation experiments to identify PLA2G4B-interacting proteins in the IL-17 pathway

• Track changes in PLA2G4B expression following experimental treatments using quantitative immunofluorescence

• Combine with siRNA knockdown studies to correlate protein reduction with functional changes

Research has demonstrated that silencing PLA2G4B with siRNA-505 inhibits keratinocyte proliferation and migration, and suppresses psoriasis progression in mouse models . The biotinylated antibody can validate successful knockdown at the protein level.

What strategies exist for investigating the JMJD7-PLA2G4B readthrough transcript?

To study the unique JMJD7-PLA2G4B readthrough transcripts:

• Epitope mapping: Compare results from antibodies targeting different regions (N-terminal vs. C-terminal) to distinguish between conventional PLA2G4B and readthrough products

• Size analysis: Western blotting can identify multiple isoforms and fusion proteins

• Tissue distribution: Quantify expression across different tissues using biotinylated antibody in flow cytometry

• Subcellular localization: Combine with organelle markers to determine compartmentalization

• Functional assays: After immunoprecipitation, assess enzymatic activity against different phospholipid substrates

The readthrough transcript encodes fusion proteins with partial JmjC domain and downstream C2 and phospholipase A2 domains, potentially exhibiting functions distinct from conventional PLA2G4B .

How can this antibody be used in multiplex immunoassays?

For multiplex experimental design:

• Utilize the biotin tag for streptavidin-based capture in multiplex bead arrays

• Combine with differently labeled antibodies against other inflammatory pathway components

• Incorporate with antibodies against IL-17 pathway proteins to investigate correlation

• Use with anti-CD1b antibodies to study the proposed lipid antigen presentation mechanism

• Employ quantum dot-streptavidin conjugates for multi-color imaging with high sensitivity

• Consider microfluidic-based approaches for analyzing multiple parameters simultaneously

When designing multiplex systems, validate that streptavidin conjugates do not interfere with other detection systems and optimize signal-to-noise ratios for each target .

What is the expected expression pattern of PLA2G4B in different human tissues?

PLA2G4B and the JMJD7-PLA2G4B readthrough transcripts show variable expression across tissues:

• Ubiquitous baseline expression: Low-level expression in most tissues

• Elevated expression in:

  • Inflammatory skin conditions (particularly psoriatic lesions)

  • Certain immune cell populations

• Subcellular localization: Primarily cytosolic, with potential membrane association during activation

• Expression changes: May be upregulated during inflammation or in response to calcium signaling

When interpreting immunoassay results, consider that different isoforms may predominate in different tissues, and readthrough products may have tissue-specific expression patterns .

How do I validate specificity when using PLA2G4B Antibody, Biotin conjugated?

To confirm antibody specificity:

• Blocking peptide experiments: Pre-incubate antibody with immunizing peptide before application

• Knockout/knockdown controls: Compare signal in PLA2G4B-silenced cells (e.g., using siRNA-505)

• Multiple antibody approach: Compare results with antibodies targeting different PLA2G4B epitopes

• Expected molecular weight verification: Confirm the ~53 kDa band in Western blots

• Positive and negative tissue controls: Use tissues with known expression patterns

• Cross-reactivity testing: Test against recombinant PLA2G4A to confirm specificity within the phospholipase family

Rigorous validation is particularly important given the complexity of readthrough transcripts and multiple isoforms .

What controls should be included when studying PLA2G4B's enzymatic activity?

When investigating PLA2G4B's phospholipase activity:

• Positive control: Include PLA2G4A (more active enzyme) as a benchmark

• Negative control: Use catalytically inactive PLA2G4B mutant

• Calcium dependency: Test activity with and without calcium (PLA2G4B is calcium-dependent)

• Substrate specificity controls: Compare activity against:

  • Arachidonoyl phospholipids (preferred substrate)

  • Palmitoyl-arachidonyl-phosphatidylethanolamine (for isoform 3)

  • Phosphatidylcholine (for isoform 5)

• Inhibitor studies: Use phospholipase inhibitors to confirm specificity of enzymatic assays

Remember that PLA2G4B has weaker enzymatic activity than PLA2G4A, and different isoforms show varying substrate specificities .