PLD6 Antibody, Biotin conjugated

What is PLD6 and why is it an important research target?

PLD6 (Phospholipase D Family, Member 6) is a multifunctional protein that serves as an endonuclease playing a critical role in PIWI-interacting RNA (piRNA) biogenesis during spermatogenesis. piRNAs provide essential protection against the activity of mobile genetic elements, thus maintaining genome stability, particularly in germline cells when transposons are mobilized during widespread genomic demethylation. PLD6 has also been proposed to function as a cardiolipin hydrolase generating phosphatidic acid at the mitochondrial surface, though this activity may be limited in vitro . This protein is also known by several synonyms including Mitochondrial cardiolipin hydrolase, Choline phosphatase 6, MitoPLD, and Protein zucchini homolog (ZUC) . Research targeting PLD6 is important for understanding germ cell development, fertility mechanisms, and mitochondrial membrane dynamics, as well as exploring its potential role in disease processes related to genome instability .

What are the key specifications of the PLD6 Antibody, Biotin conjugated?

The PLD6 Antibody, Biotin conjugated is a rabbit polyclonal antibody against human PLD6. Its key specifications include:

This comprehensive data is derived from product specifications provided by multiple antibody suppliers .

How is PLD6 structurally and functionally characterized?

PLD6 exhibits a distinctive "bell-shaped" structure comprising approximately 37.72% α-helix, 9.25% β-sheet, 43.64% random coil, and other secondary structural elements. Solvent accessibility analysis indicates that 41.36% of its amino acids have low solvent accessibility (buried), while 58.63% are exposed. Most conserved amino acids are located on the protein surface, suggesting their involvement in functional interactions .

The protein contains several highly conserved regions including Val41-Ser46 (VLFFPS), Glu91-Ser99 (ELCLFAFSS), Met151-Ala156 (MHHKFA), and Leu163-Trp170 (LITGSLNW). Subcellular localization studies place PLD6 in the outer mitochondrial membrane, with the Val10-Val32 sequence functioning as a transmembrane segment .

Functionally, Gene Ontology (GO) analysis reveals PLD6's involvement in:

• Biological processes: P granule organization, piRNA metabolism, DNA methylation, male meiosis

• Molecular functions: Phospholipase activity, catalytic hydrolysis of phosphodiester bonds

• Cellular components: Outer mitochondrial membrane, outer cell membrane, endoplasmic reticulum

During molecular dynamic simulations, PLD6 forms approximately 7-10 hydrogen bonds with cardiolipin (CL) molecules, with the aromatic ring of His-153 forming a salt bridge with phosphorus atoms in CL molecules. This binding pattern provides the structural basis for potential hydrolysis reactions .

What are the optimal experimental conditions for using PLD6 Antibody, Biotin conjugated in ELISA applications?

When designing ELISA experiments with PLD6 Antibody, Biotin conjugated, researchers should consider the following methodological approach:

• Sample preparation: For cell or tissue lysates, extract proteins using a compatible lysis buffer (RIPA or NP-40 based) supplemented with protease inhibitors. Clear lysates by centrifugation (14,000 x g for 15 minutes at 4°C).

• Coating and blocking: Coat ELISA plates with target antigen (recombinant PLD6 protein or lysates) at 1-10 μg/mL in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. Block with 3-5% BSA or 5% non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Antibody dilution: The optimal working dilution range for PLD6 Antibody, Biotin conjugated should be determined empirically, but typically start with 1:500 to 1:2000 dilution in blocking buffer. Incubate for 1-2 hours at room temperature or overnight at 4°C .

• Detection system: Utilize streptavidin-HRP conjugate (1:1000 to 1:5000 dilution) to detect the biotin-conjugated antibody. After washing extensively with PBS-T (4-5 times), add TMB substrate and measure absorbance at 450 nm after stopping the reaction with H₂SO₄.

• Controls: Include negative controls (omitting primary antibody), positive controls (using samples known to express PLD6), and concentration gradients of recombinant PLD6 for standard curve generation.

• Optimization parameters: Key parameters to titrate include antigen concentration, primary antibody dilution, streptavidin-HRP concentration, and incubation times. The optimal pH for antibody-antigen binding is typically around 7.4, which matches the provided diluent buffer .

How can PLD6 Antibody, Biotin conjugated be applied in immunohistochemistry and immunofluorescence studies?

While primarily validated for ELISA, evidence suggests PLD6 Antibody can be adapted for IHC and IF applications with appropriate protocol adjustments:

For Immunohistochemistry (IHC-P):

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Cut 4-6 μm sections and mount on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes. Based on findings with similar PLD6 antibodies, citrate buffer often yields superior results .

• Blocking and antibody application: Block endogenous peroxidase with 3% H₂O₂, then block non-specific binding with 5-10% normal serum. Apply PLD6 antibody at 1:50 to 1:200 dilution (starting at 1:100 as used successfully with related PLD6 antibodies) and incubate overnight at 4°C .

• Detection and visualization: Use biotin-streptavidin-HRP detection systems, which are particularly suitable for this biotin-conjugated antibody. Develop with DAB or AEC substrate and counterstain with hematoxylin.

For Immunofluorescence (IF/ICC):

• Cell preparation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes.

• Blocking and antibody incubation: Block with 1-5% BSA in PBS for 30-60 minutes. Apply PLD6 antibody at 1:50 to 1:200 dilution (starting with 1:100 as used with similar antibodies) and incubate overnight at 4°C .

• Detection: Use fluorophore-conjugated streptavidin (e.g., streptavidin-Alexa Fluor 488) for detection. Include appropriate counterstains such as DAPI for nuclei.

• Optimization note: When using the biotin-conjugated antibody for IF, researchers should be aware of potential endogenous biotin interference. Consider using a biotin-blocking step if background is observed .

What validation procedures should be performed when using PLD6 Antibody, Biotin conjugated in a new experimental system?

To ensure antibody specificity and reliability in a new experimental system, implement the following validation strategy:

• Positive and negative controls:

  • Positive controls: Include tissues/cells known to express PLD6 (testis tissue, HepG2 cells)

  • Negative controls: Use tissues/cells with minimal PLD6 expression or employ knockdown/knockout systems

  • Technical negative controls: Omit primary antibody while maintaining all other steps

• Western blot validation: Despite the antibody being primarily validated for ELISA, performing Western blot analysis can confirm specificity by verifying the expected molecular weight (approximately 28 kDa) . This cross-validation helps ensure the antibody recognizes the correct target.

• Dilution titration: Test a range of antibody dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine optimal signal-to-noise ratio for your specific system.

• Peptide competition assay: Pre-incubate the antibody with excess recombinant PLD6 protein before application to samples. Signal elimination/reduction confirms specificity.

• Cross-reactivity assessment: If working with non-human samples, particularly mouse tissues (where cross-reactivity is suggested but not fully validated), perform parallel experiments with human samples for comparison .

• Reproducibility testing: Perform independent replicates to ensure consistent results across multiple experiments and different sample preparations.

• Orthogonal validation: Compare results with alternative detection methods, such as mRNA expression (RT-PCR, qRT-PCR) as demonstrated in the bovine testis study .

How can PLD6 Antibody be used to investigate the relationship between PLD6 and piRNA biogenesis in spermatogenesis?

Investigating PLD6's role in piRNA biogenesis during spermatogenesis requires a multifaceted approach:

• Co-localization studies: Employ dual immunofluorescence to examine the spatial relationship between PLD6 and known piRNA pathway components (PIWI proteins, piRNA precursors) in testicular tissue sections or isolated germ cells. The biotin-conjugated PLD6 antibody can be visualized using fluorescently labeled streptavidin, while other pathway components can be detected with directly labeled antibodies.

• Developmental expression profiling: Analyze PLD6 expression patterns across different stages of spermatogenesis using immunohistochemistry on testicular sections at various developmental time points. This approach can reveal temporal correlations between PLD6 expression and key events in piRNA biogenesis, similar to the developmental analysis performed in bovine testes .

• Subcellular fractionation with immunoblotting: Isolate mitochondria, cytosolic, and nuclear fractions from testicular tissue or germline cells, then perform Western blotting to determine the subcellular distribution of PLD6. This confirms the mitochondrial outer membrane localization that is critical for its function in piRNA processing.

• Immunoprecipitation and RNA analysis: Use PLD6 antibodies for immunoprecipitation followed by RNA isolation and sequencing to identify piRNA precursors or intermediates that associate with PLD6 during processing. While the biotin-conjugated format may not be ideal for direct IP, it can be adapted using streptavidin-based capture systems.

• Translational research approach: Compare PLD6 expression and localization patterns between normal and infertile testicular samples to establish correlations between PLD6 dysfunction and reproductive pathologies.

The study of bovine testes has already demonstrated that PLD6 is expressed in all germ cells similar to the germ cell marker VASA, suggesting its importance throughout spermatogenesis . This provides a foundation for deeper investigation of its specific role in piRNA-mediated genome protection.

What are the methodological considerations when studying PLD6's proposed cardiolipin hydrolase activity using the biotin-conjugated antibody?

Investigating PLD6's potential cardiolipin hydrolase activity requires careful experimental design:

• Mitochondrial isolation and fractionation: Isolate intact mitochondria from tissues or cells, then separate outer and inner mitochondrial membranes. Use the biotin-conjugated PLD6 antibody to confirm the protein's localization to the outer mitochondrial membrane through Western blotting or immunoelectron microscopy with gold-labeled streptavidin.

• In vitro enzymatic assays: Design assays measuring phospholipase activity using purified recombinant PLD6 and cardiolipin substrates. Monitor phosphatidic acid production via thin-layer chromatography or mass spectrometry. The biotin-conjugated antibody can be used to confirm the identity and purity of immunoprecipitated PLD6 used in these assays.

• Structure-function studies: Based on molecular dynamics simulations showing that PLD6 forms approximately 7-10 hydrogen bonds with cardiolipin molecules and that the His-153 residue forms critical salt bridges , design site-directed mutagenesis experiments targeting these interaction points. The biotin-conjugated antibody can verify the expression of mutant proteins.

• Cardiolipin accessibility assessment: Since cardiolipin is predominantly located on the inner mitochondrial membrane while PLD6 resides on the outer membrane , investigate potential mechanisms that might allow interaction between these spatially separated components. Use the antibody in proximity ligation assays with cardiolipin-binding probes to assess potential contact points.

• Comparative analysis across tissues: Compare PLD6's enzymatic activity and its correlation with cardiolipin levels across different tissues. Use the biotin-conjugated antibody in combination with cardiolipin-specific dyes to assess their spatial relationship in various cell types.

• Functional validation through knockdown/overexpression: Manipulate PLD6 expression levels and measure changes in cardiolipin metabolism. The biotin-conjugated antibody can quantify PLD6 levels in these experimental systems through ELISA or Western blotting.

It's important to note that while PLD6 has been proposed to function as a cardiolipin hydrolase, research suggests this activity may be undetectable or very low in vitro , requiring highly sensitive detection methods or specific physiological conditions to be observed.

How can researchers resolve contradictory data regarding PLD6's enzymatic activities as both an endonuclease and a phospholipase?

Resolving the dual functionality paradox of PLD6 requires sophisticated experimental approaches:

• Domain-specific functional analysis: Using structural information identifying conserved regions (Val41-Ser46, Glu91-Ser99, Met151-Ala156, Leu163-Trp170) , design truncation or domain-swapping experiments to isolate regions responsible for each activity. The biotin-conjugated antibody can be used to confirm expression of these constructs if its epitope is preserved.

• Condition-dependent activity assessment: Test both endonuclease and phospholipase activities under varying biochemical conditions (pH, ion concentrations, membrane composition) to determine if these activities are conditionally regulated. Use the antibody to confirm PLD6 stability under these varied conditions.

• Conformational analysis: Investigate whether PLD6 undergoes conformational changes that might alternately expose catalytic sites for different substrates. Techniques such as hydrogen-deuterium exchange mass spectrometry can reveal structural dynamics, with the biotin-conjugated antibody serving as a capture reagent.

• Interactome mapping: Identify protein interaction partners that might direct PLD6 toward either endonuclease or phospholipase function. Proximity-dependent biotin identification (BioID) using PLD6 as bait could reveal context-specific interactions.

• Post-translational modification analysis: Examine whether post-translational modifications switch PLD6 between functional states. The biotin-conjugated antibody can be used for enrichment prior to mass spectrometry analysis.

• In vivo versus in vitro activity reconciliation: Address the discrepancy between proposed in vivo functions and limited in vitro phospholipase activity by developing more physiologically relevant assay systems. This might include reconstituted membrane systems or organelle-specific activity probes.

• Evolutionary analysis: Compare PLD6 orthologs across species to identify conserved catalytic residues for each function. The study demonstrating that bovine PLD6 groups with BIBE10 protein suggests evolutionary conservation that may provide insights into functional specialization .

By addressing these aspects systematically, researchers can determine whether PLD6's dual functionality represents true moonlighting capabilities, context-dependent specialization, or potentially misattributed activities.

What are common challenges when using PLD6 Antibody, Biotin conjugated and how can they be addressed?

Researchers may encounter several challenges when working with PLD6 Antibody, Biotin conjugated:

• High background in biotin-rich tissues:

  • Problem: Endogenous biotin in tissues like liver, kidney, and brain can cause high background.

  • Solution: Implement a biotin blocking step using streptavidin followed by free biotin before applying the primary antibody. Commercial biotin blocking kits are available and effective for IHC/IF applications.

• Signal variability across applications:

  • Problem: While validated primarily for ELISA , performance in other applications may vary.

  • Solution: Perform thorough optimization for each application, starting with conditions reported for similar PLD6 antibodies (1:100 dilution for IHC-P ). Include positive controls like testis tissue where PLD6 is consistently expressed .

• Limited cross-reactivity documentation:

  • Problem: Cross-reactivity with mouse samples is suggested but not comprehensively validated.

  • Solution: When working with non-human samples, validate antibody performance using corresponding human samples as references and confirm with complementary techniques like PCR detection of PLD6 transcript .

• Antigen masking due to protein-protein interactions:

  • Problem: PLD6's involvement in protein complexes may obscure epitopes.

  • Solution: Explore various antigen retrieval methods beyond standard citrate buffer, including EDTA-based (pH 9.0) or enzymatic retrieval approaches. Test different detergents (0.1-0.5% Triton X-100, 0.1% SDS) in sample preparation to disrupt protein interactions.

• Sensitivity to freeze-thaw cycles:

  • Problem: Repeated freeze-thaw cycles can degrade antibody performance.

  • Solution: Aliquot the antibody upon receipt and store at -20°C or -80°C . Use glycerol-containing storage buffer (as provided in the commercial formulation) to minimize freeze-thaw damage.

• Low signal intensity:

  • Problem: Insufficient signal detection especially in samples with low PLD6 expression.

  • Solution: Implement signal amplification techniques such as tyramide signal amplification (TSA) which is particularly effective with biotin-conjugated antibodies. This can enhance sensitivity by 10-100 fold while maintaining specificity.

How can researchers optimize immunohistochemical staining protocols for studying PLD6 in reproductive tissues?

Optimizing IHC protocols for PLD6 detection in reproductive tissues requires attention to several key factors:

• Tissue fixation and processing:

  • Use neutral buffered formalin (10%) for 24-48 hours depending on tissue size

  • Process tissues gently to preserve antigenic sites and cellular architecture

  • For testicular tissue, which has been successfully used to study PLD6 , consider Bouin's fixative as an alternative that preserves nuclear detail in germline cells

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods comparatively:

    • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0)

    • HIER with Tris-EDTA (pH 9.0)

    • Enzymatic retrieval with proteinase K (10 μg/mL for 10-15 minutes)

  • For each method, optimize heating time (10-30 minutes) and cooling period (20-30 minutes)

• Section thickness and handling:

  • Prepare 3-5 μm sections for optimal antibody penetration

  • Use positively charged slides and include a post-sectioning drying step (37°C overnight)

  • For testicular tissue, orientation is critical to properly visualize seminiferous tubules

• Background reduction strategies:

  • Implement dual blocking: 3% H₂O₂ followed by 5-10% normal serum

  • For reproductive tissues which may contain endogenous biotin, use commercial avidin/biotin blocking kits

  • Include 0.1-0.3% Triton X-100 in blocking buffers to reduce non-specific binding

• Antibody incubation parameters:

  • Test both short (2 hours at room temperature) and long (overnight at 4°C) incubation protocols

  • Optimize antibody concentration through dilution series (1:50, 1:100, 1:200, 1:500)

  • Use humidity chambers to prevent section drying and maintain consistent antibody concentration

• Specialized detection strategies:

  • For multiplexing with other markers (such as VASA or UCHL1 as used in bovine studies ), use sequential detection protocols with careful antibody stripping between rounds

  • Consider enzyme-labeled streptavidin (HRP or AP) followed by substrate development for bright-field imaging

  • For fluorescence applications, select fluorophore-conjugated streptavidin with spectra distinct from other fluorescent probes used in multi-labeling experiments

• Counterstaining optimization:

  • For reproductive tissues, customize hematoxylin counterstaining time (30 seconds to 3 minutes) to achieve optimal nuclear detail without obscuring specific staining

  • Consider using DAPI or other nuclear counterstains that allow clear visualization of different stages of spermatogenesis

What are the best practices for quantifying PLD6 expression in different cell types using the biotin-conjugated antibody?

Accurate quantification of PLD6 expression across cell types requires rigorous methodological approaches:

• Sample preparation standardization:

  • Standardize cell numbers, protein extraction methods, and protein quantification techniques

  • For tissue samples, consider laser capture microdissection to isolate specific cell populations (e.g., specific stages of spermatogenic cells)

  • Prepare standard curves using recombinant PLD6 protein for absolute quantification

• Quantitative ELISA development:

  • Develop a sandwich ELISA using a capture antibody against a different epitope of PLD6 and the biotin-conjugated antibody as detection reagent

  • Generate standard curves using recombinant PLD6 protein at concentrations spanning the physiological range (typically 0.1-100 ng/mL)

  • Include internal controls from cell lines with known PLD6 expression levels in each assay

• Image-based quantification for IHC/IF:

  • Use automated image analysis software with consistent thresholding parameters

  • Quantify staining intensity using integrated optical density (IOD) measurements

  • For cellular heterogeneity, implement cell-type identification algorithms based on morphological features or co-staining with cell-type-specific markers

  • Express results as percentage of positive cells and mean staining intensity

• Multi-parameter flow cytometry:

  • For suspension cells or dissociated tissues, develop flow cytometry protocols using the biotin-conjugated antibody with fluorescently-labeled streptavidin

  • Include cell surface markers to distinguish cell populations

  • For intracellular PLD6 detection, optimize fixation and permeabilization conditions

• Normalization strategies:

  • Normalize expression to housekeeping proteins (β-actin, GAPDH) for Western blot analysis

  • For tissue sections, normalize to tissue area or cell count

  • In heterogeneous samples like testis, normalize to specific cell type markers rather than total protein

• Statistical approach:

  • Collect data from multiple biological replicates (minimum n=3)

  • Apply appropriate statistical tests (ANOVA for multiple comparisons, t-tests for pairwise comparisons)

  • Report both means and measures of variability (standard deviation or standard error)

  • Consider using non-parametric tests for data that doesn't follow normal distribution

• Validation with orthogonal methods:

  • Correlate protein expression data with mRNA levels from qRT-PCR

  • Consider proteomics approaches for independent verification

  • As demonstrated in bovine testis research, complement antibody-based detection with transcript analysis

How might PLD6 Antibody, Biotin conjugated be used to investigate connections between mitochondrial function and piRNA biogenesis?

The dual roles of PLD6 in mitochondrial function and piRNA processing present unique research opportunities:

• Mitochondrial dynamics and piRNA processing:

  • Use the biotin-conjugated PLD6 antibody in combination with mitochondrial morphology markers to investigate whether changes in mitochondrial fusion/fission correlate with piRNA production

  • Develop live-cell imaging approaches using the antibody to visualize PLD6 localization during mitochondrial remodeling events

  • Isolate mitochondria at different functional states and quantify associated PLD6 levels and activity

• Metabolic regulation of piRNA biogenesis:

  • Investigate whether metabolic stress conditions that alter mitochondrial function affect PLD6 localization and activity

  • Use the antibody to track PLD6 redistribution under conditions like oxidative stress, nutrient deprivation, or hypoxia

  • Correlate changes in PLD6 localization/expression with piRNA profiles under different metabolic conditions

• Mitochondrial-nuclear communication:

  • Explore if PLD6's role in generating phosphatidic acid at the mitochondrial surface serves as a signaling mechanism affecting nuclear processes including piRNA gene transcription

  • Use the antibody in proximity ligation assays to detect interactions between PLD6 and components of signaling pathways

  • Develop co-immunoprecipitation protocols using streptavidin capture of the biotin-conjugated antibody to identify novel interaction partners

• Developmental coordination:

  • Examine whether mitochondrial maturation during germ cell development correlates with changes in PLD6 expression and localization

  • Use the antibody in developmental time-course studies of testicular tissue

  • Apply super-resolution microscopy with the biotin-conjugated antibody to visualize potential changes in PLD6 distribution patterns during cellular differentiation

• Pathological connections:

  • Investigate PLD6 expression and localization in models of mitochondrial dysfunction, male infertility, or genomic instability

  • Develop therapeutic approaches targeting PLD6 and assess efficacy using the antibody as a pharmacodynamic marker

The "bell-shaped" structural configuration of PLD6 identified through molecular modeling may provide insights into how the protein can perform dual functions, with specific domains potentially dedicated to either mitochondrial interaction or piRNA processing. The biotin-conjugated antibody could be used in epitope mapping studies to further characterize these functional domains.

What innovative approaches could integrate PLD6 Antibody, Biotin conjugated with emerging technologies for reproductive biology research?

Integration of PLD6 antibody with cutting-edge technologies opens new research avenues:

• Single-cell protein profiling:

  • Adapt the biotin-conjugated antibody for mass cytometry (CyTOF) to simultaneously analyze PLD6 expression alongside dozens of other proteins at single-cell resolution

  • Develop microfluidic-based single-cell Western blotting protocols using the antibody to examine PLD6 heterogeneity in rare cell populations

  • Incorporate the antibody into multiplexed ion beam imaging (MIBI) workflows for subcellular spatial resolution of PLD6 in tissue contexts

• Spatial transcriptomics integration:

  • Combine immunofluorescence using the biotin-conjugated antibody with in situ sequencing or spatial transcriptomics to correlate PLD6 protein localization with transcriptional states

  • Develop sequential immunofluorescence and RNA-FISH protocols to visualize PLD6 protein alongside piRNA precursors

  • Create computational pipelines that integrate protein expression data with spatial transcriptome information

• Organoid and advanced culture systems:

  • Apply the antibody to testicular or germline organoids to track PLD6 expression during in vitro gametogenesis

  • Use microfluidic testis-on-a-chip systems with real-time imaging capabilities and the biotin-conjugated antibody to monitor PLD6 dynamics during spermatogenesis

  • Develop differentiation protocols from pluripotent stem cells to germline lineages using PLD6 as a differentiation marker

• CRISPR-based functional genomics:

  • Implement CRISPR activation/inhibition screens targeting genes in the PLD6 pathway, using the antibody to quantify resulting changes in PLD6 expression

  • Develop CRISPR base editing approaches to introduce specific mutations in the conserved regions of PLD6 identified through structural studies

  • Create CRISPR knock-in reporter systems where PLD6 is tagged with a fluorescent protein, using the antibody to validate proper targeting

• Artificial intelligence applications:

  • Train deep learning algorithms on immunofluorescence images using the biotin-conjugated antibody to automatically identify and classify cell types based on PLD6 expression patterns

  • Develop predictive models correlating PLD6 expression patterns with fertility outcomes

  • Use machine learning to analyze large datasets integrating PLD6 protein levels with transcriptomics and clinical parameters

These innovative approaches could provide unprecedented insights into PLD6's role in reproductive biology, potentially leading to new diagnostic tools or therapeutic strategies for fertility disorders associated with defects in piRNA biogenesis or mitochondrial function.