PLD5 Antibody

What is PLD5 and why is it significant in research?

PLD5 (Phospholipase D Family, Member 5) is a protein categorized within the phospholipase D family, though interestingly it is considered an inactive phospholipase D5 protein . Despite being part of this enzyme family, the specific function of PLD5 remains largely unknown in current literature . The protein has a molecular weight of approximately 49kDa and consists of 444 amino acids in one of its isoforms . Research interest in PLD5 stems from its potential involvement in cellular signaling pathways, though its precise biological role requires further elucidation. The protein is encoded by the gene located at NCBI Gene ID 200150, with alias symbol PLDC . Its study is facilitated through the use of specific antibodies that recognize different epitopes of the protein, enabling researchers to investigate its expression, localization, and potential functions.

What epitopes of PLD5 are commonly targeted by research antibodies?

Several distinct epitopes of PLD5 are targeted by commercially available antibodies, with particular emphasis on different regions of the protein:

• C-terminal region antibodies: Multiple manufacturers offer antibodies targeting the C-terminal portions of PLD5, specifically amino acids 508-536 and 515-544 . These C-terminal antibodies are frequently used in Western blotting, immunofluorescence, and immunohistochemistry applications.

• Middle region antibodies: Antibodies recognizing amino acids 92-536 and 189-238 are also available, providing coverage of central regions of the protein.

• Specific peptide-targeted antibodies: Some manufacturers use synthetic peptides as immunogens, such as the sequence "SSLKAICTEIANCSLKVKFFDLERENACATKEQKNHTFPRLNRNKYMVTD" or "AGTGLVINQADVRNNRSIIKQLKDVFERDWYSPYAKTLQPTKQPNCSSLF" .

The choice of epitope can significantly impact experimental outcomes, as different regions may be more accessible depending on protein folding, post-translational modifications, or protein-protein interactions present in experimental conditions.

How do polyclonal and monoclonal PLD5 antibodies differ in research applications?

Most commercially available PLD5 antibodies are polyclonal antibodies raised in rabbits, as evidenced by the information from multiple suppliers . Polyclonal antibodies offer several advantages in PLD5 research:

• Recognition of multiple epitopes: Polyclonal antibodies bind to various epitopes on the PLD5 protein, potentially increasing detection sensitivity, especially in applications where protein conformation may vary.

• Robust signal detection: The ability to bind multiple epitopes can enhance signal strength in applications like Western blotting and immunohistochemistry.

• Tolerance to minor protein denaturation: Polyclonal antibodies typically maintain reactivity even if some epitopes are modified during experimental procedures.

What are the primary applications for PLD5 antibodies in research?

PLD5 antibodies support various research applications as evidenced by manufacturer specifications:

When designing experiments, researchers should select antibodies validated specifically for their application of interest, as performance can vary considerably across different experimental contexts.

What factors should guide selection of a PLD5 antibody for specific research applications?

When selecting a PLD5 antibody for research, several critical factors should be evaluated:

• Species reactivity: Consider the experimental model organism. Different PLD5 antibodies show varying reactivity profiles across species. For example, some antibodies react primarily with human PLD5 , while others exhibit cross-reactivity with multiple species including mouse, rat, bovine, and others .

• Epitope location: The antibody's target region may influence detection capabilities in different applications. C-terminal antibodies (e.g., targeting amino acids 508-536) may perform differently than those targeting middle regions (e.g., amino acids 92-536) .

• Conjugation requirements: Depending on the application, researchers may need unconjugated antibodies or those conjugated to specific molecules:

  • HRP conjugation for enhanced colorimetric detection in ELISA and Western blotting

  • FITC conjugation for fluorescence applications

  • Biotin conjugation for avidin-based detection systems

• Validation data: Review available validation data for the specific application. Antibodies validated for Western blotting may not necessarily perform optimally in immunohistochemistry.

• Homology considerations: For cross-species studies, evaluate the predicted homology based on immunogen sequence. For example, one PLD5 antibody shows 100% homology to human, 86% to mouse, 92% to rat, and varying degrees to other species .

How should PLD5 antibodies be validated prior to use in critical experiments?

Thorough validation of PLD5 antibodies is essential before employing them in critical research. A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive controls: Use tissues or cell lines known to express PLD5

  • Negative controls: Include samples where PLD5 is absent or knocked down

  • Isotype controls: Use matched isotype antibodies to control for non-specific binding

• Blocking peptide competition assays: Some manufacturers offer blocking peptides (e.g., AAP67749 for the ARP67749_P050 antibody) . These peptides, containing the immunogen sequence, can be used to confirm signal specificity by pre-incubating with the antibody before application to samples.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of PLD5 to confirm consistent results.

• Western blot validation: Confirm the antibody detects a band of appropriate molecular weight (approximately 49kDa for certain isoforms) .

• Dilution optimization: Test multiple antibody dilutions to determine optimal working concentration, as manufacturers often recommend that "optimal working dilution should be determined by the investigator" .

• Signal specificity verification: Particularly for newer or less-characterized antibodies, additional verification through genetic approaches (siRNA knockdown, CRISPR knockout) provides robust validation.

What are the optimal storage and handling conditions for PLD5 antibodies?

Proper storage and handling of PLD5 antibodies are crucial for maintaining reactivity and specificity:

• Short-term storage: Most PLD5 antibodies can be stored at 2-8°C for short periods (up to 1-2 weeks) .

• Long-term storage: For extended storage, maintain antibodies at -20°C in small aliquots to prevent freeze-thaw cycles . Repeated freeze-thaw cycles can significantly diminish antibody performance.

• Buffer conditions: Most commercial PLD5 antibodies are supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose , which helps stabilize the antibody proteins.

• Aliquoting strategy: Upon receipt, dividing the antibody into single-use aliquots minimizes degradation from repeated freeze-thaw cycles.

• Handling precautions: When working with conjugated antibodies (HRP, FITC, or biotin), additional care should be taken to protect from light exposure (especially for fluorophore-conjugated antibodies) and avoid contamination.

• Expiration considerations: Most commercial PLD5 antibodies have a shelf life of approximately 12 months from receipt when stored properly .

What are common causes of non-specific binding when using PLD5 antibodies?

Non-specific binding is a frequent challenge when working with PLD5 antibodies. Several factors can contribute to this issue:

• Antibody concentration: Excessive antibody concentration often increases background signal. Optimization through titration experiments is recommended, as manufacturers note that "optimal working dilution should be determined by the investigator" .

• Blocking conditions: Inadequate blocking can lead to non-specific binding. Optimize blocking agent (BSA, normal serum, commercial blockers) concentration and incubation time based on the specific application.

• Cross-reactivity: Some PLD5 antibodies may cross-react with related proteins. For example, antibodies targeting conserved domains might recognize other phospholipase family members. Evaluate predicted cross-reactivity based on epitope sequence conservation.

• Sample preparation: Incomplete fixation or improper antigen retrieval can contribute to non-specific binding in immunohistochemistry and immunocytochemistry applications.

• Secondary antibody issues: Non-specific binding of secondary antibodies can be addressed by using more highly cross-absorbed secondary antibodies or pre-adsorption steps.

To address these issues, researchers should implement appropriate controls, including isotype controls, secondary-only controls, and blocking peptide competition assays using available blocking peptides (such as AAP67749 for specific antibodies) .

How can inconsistent Western blot results with PLD5 antibodies be resolved?

Inconsistent Western blotting results when using PLD5 antibodies may stem from several sources:

• Sample preparation variations:

  • Standardize protein extraction methods

  • Ensure consistent protein denaturation conditions

  • Verify equal loading using housekeeping protein controls

• Transfer efficiency issues:

  • Optimize transfer conditions for high molecular weight proteins

  • Confirm transfer efficiency using reversible staining methods

• Antibody-specific factors:

  • Verify antibody is detecting the correct molecular weight (approximately 49kDa for certain isoforms)

  • Consider that PLD5 may have multiple isoforms or post-translational modifications affecting migration patterns

  • Test different antibody lots if inconsistency correlates with lot changes

• Protocol optimization:

  • Adjust blocking conditions to reduce background

  • Optimize primary antibody concentration and incubation time/temperature

  • Evaluate different detection systems (chemiluminescence vs. fluorescence)

• Experimental controls:

  • Include positive control lysates from tissues/cells known to express PLD5

  • Consider using blocking peptides to confirm specificity of bands

Systematic troubleshooting with controlled variables and proper documentation will help identify the source of inconsistency in Western blotting results.

What controls are essential when using PLD5 antibodies in immunohistochemistry?

When performing immunohistochemistry with PLD5 antibodies, several controls are critical for result interpretation:

• Positive tissue controls:

  • Include tissues with known PLD5 expression

  • Verify staining patterns match expected cellular localization

• Negative controls:

  • Omit primary antibody but include all other reagents

  • Use isotype-matched control antibodies

  • When available, include tissues known to lack PLD5 expression

• Antibody validation controls:

  • Pre-absorption with blocking peptides where available

  • Comparison with alternative PLD5 antibodies targeting different epitopes

• Technical controls:

  • Include serial dilutions of primary antibody to demonstrate specificity

  • Perform antigen retrieval optimization

  • Include internal control tissues within sections when possible

• Specificity verification:

  • Compare staining patterns with published literature or database resources like The Human Protein Atlas

  • Consider orthogonal validation using in situ hybridization for PLD5 mRNA

Well-designed controls not only validate experimental results but also provide critical information for troubleshooting when unexpected staining patterns emerge.

How does PLD5 antibody epitope selection impact protein-protein interaction studies?

The choice of PLD5 antibody epitope can significantly influence protein-protein interaction (PPI) studies through several mechanisms:

• Epitope masking: PLD5 interacts with other proteins, including UBC (ubiquitin C) . Antibodies targeting regions involved in these interactions may be unable to bind PLD5 when it is engaged in protein complexes, potentially leading to false-negative results in co-immunoprecipitation studies.

• Interaction domain considerations: When studying PPIs, selecting antibodies that target regions away from known or predicted interaction domains can minimize interference. For example, if the C-terminal region is involved in protein interactions, antibodies targeting amino acids 92-536 might be more suitable for detecting complexes.

• Conformation-sensitive detection: Some antibodies may preferentially recognize specific conformational states of PLD5. This property can be leveraged to study how protein interactions affect PLD5 structure by using conformation-sensitive antibodies.

• Cross-linking compatibility: For methods involving chemical cross-linking, antibodies whose epitopes contain reactive amino acids (lysine, cysteine) may show reduced binding after cross-linking procedures.

• Validation strategies: When investigating novel PPIs, validating findings using multiple antibodies targeting different PLD5 epitopes strengthens confidence in results and controls for epitope-specific artifacts.

What approaches are recommended for studying PLD5 expression in neurological disease models?

Given the potential neurological relevance of PLD5, several methodological approaches are recommended when studying this protein in neurological disease contexts:

• Multi-method expression analysis:

  • Western blotting for total protein quantification

  • Immunohistochemistry for spatial distribution analysis

  • qPCR for transcript level comparison with protein expression

• Cell-type specific analysis:

  • Double immunofluorescence with cell-type markers (neurons, glia, etc.)

  • Single-cell analysis techniques compatible with PLD5 antibodies

  • Laser capture microdissection combined with Western blotting

• Subcellular localization:

  • Fractionation studies with Western blotting

  • High-resolution microscopy with PLD5 antibodies

  • Co-localization with organelle markers

• Disease model considerations:

  • Temporal expression analysis across disease progression

  • Comparison between affected and unaffected brain regions

  • Correlation with disease-specific markers or pathologies

• Human tissue validation:

  • Translation of findings from models to human samples

  • Consideration of post-mortem protein degradation effects

  • Coordination with brain bank resources and clinical data

For these studies, antibodies with demonstrated reactivity in the appropriate species (human, mouse, rat) should be selected, with careful attention to validation in neural tissues.

How can PLD5 antibodies be employed in studying post-translational modifications?

Investigating post-translational modifications (PTMs) of PLD5 requires specialized approaches:

• PTM-specific antibodies:

  • While the search results don't mention PTM-specific antibodies for PLD5, researchers can explore developing custom antibodies against predicted modification sites

  • Phosphorylation, ubiquitination, and glycosylation are common PTMs that might regulate PLD5 function

• Complementary techniques:

  • Immunoprecipitation with general PLD5 antibodies followed by PTM-specific Western blotting

  • Mass spectrometry analysis of immunoprecipitated PLD5 to identify modifications

  • In vitro modification assays combined with antibody detection

• Epitope considerations:

  • Select PLD5 antibodies targeting regions distant from potential modification sites to avoid interference

  • Compare binding efficiency of different antibodies before and after treatments that alter PTM status

• Experimental design:

  • Include appropriate controls for PTM-inducing conditions

  • Employ PTM-blocking or -enhancing treatments to validate specificity

  • Consider the impact of protein conformation changes on antibody accessibility

• Technical challenges:

  • PTMs may alter protein migration in SDS-PAGE, requiring careful interpretation of Western blot results

  • Low abundance of modified forms may necessitate enrichment strategies

  • Transient modifications may require rapid sample processing to preserve PTM status

What emerging techniques can be combined with PLD5 antibodies for advanced subcellular localization studies?

Several cutting-edge techniques can be paired with PLD5 antibodies to achieve high-resolution subcellular localization data:

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) microscopy with fluorophore-conjugated PLD5 antibodies

  • STORM/PALM approaches for single-molecule localization

  • Expansion microscopy to physically enlarge specimens labeled with PLD5 antibodies

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins combined with PLD5 antibody validation

  • Proximity ligation assay (PLA) to detect PLD5 interactions with potential partners

  • HRP-conjugated PLD5 antibodies for APEX-like proximity labeling

• Live-cell approaches:

  • Correlative light-electron microscopy using PLD5 antibodies

  • Intrabody development based on characterized PLD5 antibody binding sites

  • Quantitative time-lapse imaging with tagged PLD5 validated by antibody studies

• Tissue-specific applications:

  • Cleared tissue microscopy with PLD5 antibodies for 3D visualization

  • Highly multiplexed imaging (CycIF, CODEX) including PLD5 antibodies

  • Spatial transcriptomics correlated with PLD5 protein localization

• Functional localization:

  • Local proteomics approaches validated by PLD5 antibody colocalization

  • Optogenetic or chemogenetic perturbation with antibody-based readouts

  • Trafficking studies using pulse-chase approaches with surface PLD5 antibodies

These advanced approaches require careful optimization and validation of antibody specificity in each experimental context, ideally using multiple antibodies targeting different epitopes of PLD5.

What are the current limitations in PLD5 antibody research that need to be addressed?

Despite the availability of various commercial PLD5 antibodies, several limitations persist:

• Functional understanding gaps: The function of PLD5 protein remains largely unknown , which complicates interpretation of antibody-based studies and validation strategies.

• Isoform specificity: Available antibodies may not distinguish between potential PLD5 isoforms, necessitating careful experimental design when studying specific variants.

• Validation depth: While antibodies are tested for applications like Western blotting, ELISA, and immunohistochemistry , the depth of validation varies, and knockout validation data appears limited in the search results.

• Cross-reactivity documentation: Although predicted reactivity across species is provided for some antibodies , systematic validation across these species is often limited.

• Application optimization: Detailed protocols and optimization strategies specific to PLD5 antibodies are generally lacking, with manufacturers noting that "optimal working dilution should be determined by the investigator" .

Future research should address these limitations through more comprehensive validation, detailed application-specific protocols, and correlation with emerging functional data about PLD5 biology.

How might advances in antibody technology influence future PLD5 research?

Emerging antibody technologies hold promise for advancing PLD5 research:

• Recombinant antibody development: Moving from polyclonal to recombinant monoclonal antibodies could enhance reproducibility and specificity in PLD5 studies.

• Nanobodies and single-domain antibodies: These smaller binding molecules may access epitopes unavailable to conventional antibodies, potentially revealing new insights about PLD5 structure and interactions.

• Intrabodies and targeted protein degradation: Cell-permeable antibody derivatives could enable acute manipulation of PLD5 in living cells, complementing genetic approaches.

• Spatially-resolved antibody-based proteomics: Technologies like Digital Spatial Profiling could provide detailed information about PLD5 expression in complex tissues while preserving spatial context.

• Multiparametric antibody applications: Highly multiplexed imaging and flow cytometry incorporating PLD5 antibodies could reveal complex relationships between PLD5 and other proteins across diverse cell states.

These technological advances, coupled with increasing functional understanding of PLD5 biology, promise to expand the utility of PLD5 antibodies as research tools in both basic science and translational research contexts.