ppk3 Antibody

What is PRICKLE3 protein and why is it relevant to study?

PRICKLE3 (Prickle-like protein 3) is a critical protein involved in the planar cell polarity (PCP) pathway, which is essential for the polarization of epithelial cells during morphogenetic processes, including gastrulation and neurulation. It functions as part of the core module in the PCP pathway, where distinct complexes segregate to opposite sides of the cell and interact with neighboring cells at or near adherent junctions. Additionally, PRICKLE3 plays significant roles in basal body organization and cilia growth and positioning. Understanding PRICKLE3 function provides insights into cellular organization and potential disease mechanisms, making it an important target for developmental biology and cancer research .

What are the key characteristics of the PRICKLE3 Antibody (PACO61959)?

The PRICKLE3 Antibody (PACO61959) is a rabbit-derived polyclonal antibody designed for specific detection of human Prickle-like protein 3. It was generated using a peptide sequence from human PRICKLE3 protein (amino acids 9-24) as the immunogen. This antibody is delivered in liquid form (50μl) and is preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. It has been affinity-purified and validated for multiple applications including Western blot (WB), immunohistochemistry (IHC), and ELISA, with recommended dilution ranges specific to each technique. Western blot analysis has confirmed its ability to detect PRICKLE3 at the predicted molecular weight of 69 kDa in multiple human cell lines including HeLa, Jurkat, and PC-3 .

What experimental applications has the PRICKLE3 antibody been validated for?

The PRICKLE3 antibody (PACO61959) has been validated for three primary experimental applications:

• Western Blot (WB): Successfully detects PRICKLE3 in whole cell lysates from HeLa, Jurkat, and PC-3 cell lines at a recommended dilution range of 1:1000-1:5000. Optimal results were observed at 1:2000 dilution when using a goat anti-rabbit IgG secondary antibody at 1:50000 dilution. The antibody detects the target protein at 69 kDa.

• Immunohistochemistry (IHC): Effectively stains PRICKLE3 in paraffin-embedded human heart tissue at dilutions between 1:20-1:200, with optimal staining observed at 1:100. The protocol involves dewaxing, hydration, antigen retrieval in citrate buffer (pH 6.0), blocking with 10% normal goat serum, and overnight incubation with primary antibody at 4°C.

• ELISA: Validated at dilution ranges of 1:2000-1:10000, though specific protocol details were not provided in the available data .

What is the molecular structure and function of the target PRICKLE3 protein?

PRICKLE3 (UniProt Code: O43900) is a 59,097 Da protein characterized by multiple LIM domains. The LIM domain is a cysteine-rich sequence motif that binds zinc atoms to form a specific protein-binding interface, facilitating protein-protein interactions. PRICKLE3 contains three such LIM domains, giving it the alternative name "Triple LIM domain protein 6." Functionally, it participates in the planar cell polarity pathway, which regulates the coordinated polarization of cells within tissue planes. PRICKLE3 is part of the core module of this pathway and helps establish cell asymmetry by localizing to specific cellular compartments. Beyond its role in planar cell polarity, PRICKLE3 contributes to basal body organization and cilia growth and positioning. The protein is encoded on chromosome Xp11.23 and has several known synonyms including LMO6, LMO-6, Pk3, and Prickle planar cell polarity protein 3 .

How should PRICKLE3 antibody be optimized for Western blot applications?

For optimal Western blot results with PRICKLE3 antibody (PACO61959), researchers should follow these methodological guidelines:

• Sample Preparation: Prepare whole cell lysates from appropriate human cell lines (validated options include HeLa, Jurkat, and PC-3 cells). Cell lysis should be performed using standardized protocols that preserve protein integrity.

• Protein Separation: Load 20-50 μg of protein per lane on a 10-12% SDS-PAGE gel for optimal separation around the expected molecular weight of 69 kDa.

• Transfer and Blocking: After electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane using standard protocols. Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Antibody Dilution: Dilute the PRICKLE3 antibody at 1:2000 in blocking buffer (the validated optimal dilution), though the working range is 1:1000-1:5000. Incubate the membrane with diluted primary antibody overnight at 4°C with gentle agitation.

• Secondary Antibody: Use goat anti-rabbit IgG conjugated with HRP at 1:50000 dilution for detection. Incubate for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence (ECL) substrate. The expected band size is 69 kDa, though a predicted alternative band at 60 kDa may also be observed.

• Controls: Include appropriate positive controls (HeLa, Jurkat, or PC-3 cell lysates) and negative controls to validate specificity .

What protocol modifications are necessary for effective immunohistochemistry with PRICKLE3 antibody?

For effective immunohistochemistry using PRICKLE3 antibody (PACO61959), the following protocol modifications and optimization steps are recommended:

• Tissue Preparation: Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thick). Human heart tissue has been validated, but other tissues expressing PRICKLE3 may also be suitable.

• Deparaffinization and Rehydration: Perform standard dewaxing and hydration steps using xylene and graded alcohols.

• Antigen Retrieval: Critical for optimal staining, perform heat-induced epitope retrieval using citrate buffer (pH 6.0) under high pressure. This step significantly improves antibody accessibility to the target epitope.

• Blocking Endogenous Activity: Block endogenous peroxidase activity with 3% hydrogen peroxide followed by protein blocking with 10% normal goat serum for 30 minutes at room temperature.

• Primary Antibody Dilution: Prepare PRICKLE3 antibody at a dilution of 1:100 (optimal) in 1% BSA. The working range is 1:20-1:200, but titration may be necessary for different tissue types. Incubate sections overnight at 4°C in a humidified chamber.

• Detection System: Use a biotinylated secondary antibody followed by an HRP-conjugated streptavidin-biotin (SP) detection system for optimal signal amplification. Automated staining systems (such as the Leica BondTM system) can provide consistent results.

• Counterstaining: Apply hematoxylin counterstaining for nuclear visualization, followed by dehydration and mounting with a permanent mounting medium.

• Controls: Include appropriate positive control tissues (human heart) and negative controls (primary antibody omission) to validate staining specificity .

How can researchers validate the specificity of PRICKLE3 antibody in their experimental system?

To rigorously validate the specificity of PRICKLE3 antibody in experimental systems, researchers should implement the following methodological approaches:

• Genetic Validation:

  • Utilize PRICKLE3 knockout (KO) cell lines or tissues as negative controls

  • Compare antibody signal between wild-type and KO samples using identical conditions

  • Employ PRICKLE3 overexpression systems as positive controls

• Molecular Weight Verification:

  • Confirm that the detected band appears at the expected molecular weight (69 kDa for PRICKLE3)

  • Be aware of potential splice variants or post-translational modifications that may alter the observed molecular weight

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (PRICKLE3 peptide 9-24AA)

  • Run parallel experiments with and without peptide competition

  • Specific signals should be significantly reduced or eliminated in the presence of competing peptide

• Cross-reactivity Assessment:

  • Test antibody reactivity against related proteins (other PRICKLE family members)

  • Examine species cross-reactivity if working with non-human models (note that PACO61959 is validated for human samples)

• Orthogonal Detection Methods:

  • Confirm PRICKLE3 expression using alternative antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression (RT-PCR or RNA-seq)

  • Consider mass spectrometry validation of immunoprecipitated proteins

• Application-specific Controls:

  • For IHC: Include isotype controls and secondary-only controls

  • For WB: Include loading controls and molecular weight markers

What are the recommended storage and handling procedures to maintain PRICKLE3 antibody activity?

To maintain optimal PRICKLE3 antibody activity and prevent degradation, researchers should adhere to these storage and handling recommendations:

• Storage Temperature: Store the antibody at -20°C for long-term preservation. The antibody formulation contains 50% glycerol to prevent freeze-thaw damage.

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to minimize freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity by approximately 10%.

• Working Solution Handling: When preparing working dilutions, use freshly prepared buffers. Keep diluted antibody on ice during experiment preparation and use within 24 hours.

• Buffer Composition: The antibody is supplied in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. When diluting, maintain buffer compatibility by using TBST or PBST with 1-5% BSA or non-fat milk.

• Contamination Prevention: Use sterile technique when handling the antibody. Consider adding sodium azide (0.02%) to diluted antibody solutions if they need to be stored for short periods (not recommended for HRP-based detection systems).

• Temperature Transitions: Allow the antibody to equilibrate to room temperature before opening the vial to prevent condensation that could promote microbial growth.

• Expiration Monitoring: Track the production and expiration dates. While properly stored antibodies may retain activity beyond the stated expiration date, critical experiments should use antibodies within their validated shelf life.

• Transport Conditions: If transporting between laboratories, maintain cold chain using dry ice or cooling packs .

How can PRICKLE3 antibody be used to investigate planar cell polarity mechanisms in development and disease?

PRICKLE3 antibody offers valuable approaches for investigating planar cell polarity (PCP) mechanisms in developmental processes and disease states:

• Subcellular Localization Studies:

  • Employ immunofluorescence with PRICKLE3 antibody to map its asymmetric distribution within polarized cells

  • Co-localize PRICKLE3 with other PCP components (such as VANGL, CELSR, or DISHEVELLED proteins) to elucidate complex formation dynamics

  • Analyze PRICKLE3 localization at different developmental stages to track temporal changes in PCP establishment

• Tissue Architecture Analysis:

  • Use IHC with PRICKLE3 antibody on tissue sections from developmental series to correlate protein expression with morphogenetic events

  • Investigate potential alterations in PRICKLE3 expression or localization in congenital disorders involving tissue polarity defects

  • Compare PRICKLE3 distribution in normal versus pathological tissue samples (particularly in epithelial cancers where polarity is disrupted)

• Molecular Complex Identification:

  • Perform co-immunoprecipitation using PRICKLE3 antibody to identify novel binding partners in the PCP pathway

  • Combine with mass spectrometry analysis to characterize the complete PRICKLE3 interactome

  • Validate interactions through reciprocal co-IP and proximity ligation assays

• Functional Perturbation Studies:

  • Correlate PRICKLE3 expression levels (detected via Western blot) with phenotypic outcomes in knockdown/knockout models

  • Assess how mutations in PRICKLE3 or other PCP components affect PRICKLE3 localization and function

  • Examine PRICKLE3 dynamics during induced polarity disruption (using cytoskeletal inhibitors or genetic perturbations)

• Ciliary Function Investigation:

  • Study PRICKLE3 localization relative to basal bodies and ciliary structures

  • Investigate the role of PRICKLE3 in ciliopathies using patient-derived samples

  • Analyze how PRICKLE3 contributes to cilia-dependent signaling pathways in development

What approaches can be used to study PRICKLE3 interaction with other proteins in the planar cell polarity pathway?

To comprehensively investigate PRICKLE3 interactions with other proteins in the planar cell polarity pathway, researchers can employ these sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP) Strategies:

  • Perform forward Co-IP using PRICKLE3 antibody to pull down protein complexes from cell lysates

  • Conduct reverse Co-IP using antibodies against known or suspected PCP pathway proteins

  • Implement stringency gradients in washing buffers to identify both strong and transient interactions

  • Use crosslinking reagents to stabilize weak or transient interactions before immunoprecipitation

• Proximity-based Interaction Assays:

  • Apply proximity ligation assay (PLA) to visualize PRICKLE3 interactions with other proteins in situ with spatial resolution below 40 nm

  • Employ BioID or TurboID approaches by fusing biotin ligase to PRICKLE3 to identify proximal proteins

  • Utilize FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to study dynamic interactions in live cells

• Domain-specific Interaction Mapping:

  • Generate truncated PRICKLE3 constructs to map which domains (particularly the LIM domains) mediate specific protein interactions

  • Use peptide arrays to identify specific binding motifs within PRICKLE3 that facilitate protein-protein interactions

  • Perform site-directed mutagenesis of key residues to validate interaction interfaces

• Functional Validation of Interactions:

  • Implement siRNA knockdown or CRISPR-Cas9 knockout of interaction partners to assess effects on PRICKLE3 localization and function

  • Develop competition assays with peptide mimetics to disrupt specific interactions

  • Create reporter systems to quantitatively measure interaction strength under various cellular conditions

• Structural Biology Approaches:

  • Purify PRICKLE3 protein complexes for structural determination using X-ray crystallography or cryo-electron microscopy

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

  • Use NMR spectroscopy to characterize dynamic interactions between PRICKLE3 and partner proteins

How can researchers differentiate between PRICKLE3 and other PRICKLE family members in their experiments?

Differentiating between PRICKLE3 and other PRICKLE family members (PRICKLE1, PRICKLE2, and PRICKLE4) requires careful experimental design and validation:

• Antibody Epitope Selection and Validation:

  • The PRICKLE3 antibody (PACO61959) targets a unique peptide sequence (amino acids 9-24) that differs from other PRICKLE family members

  • Conduct sequence alignment analysis to identify regions of high divergence between PRICKLE paralogs

  • Perform western blot analysis across cell lines with known differential expression of PRICKLE family members to confirm specificity

  • Consider using epitope-tagged versions of different PRICKLE proteins as positive controls

• Molecular Weight Discrimination:

  • PRICKLE3 has a molecular weight of approximately 59 kDa (observed at 69 kDa on SDS-PAGE)

  • PRICKLE1 (~95 kDa), PRICKLE2 (~93 kDa), and PRICKLE4 (~40 kDa) have distinct molecular weights

  • Use high-resolution SDS-PAGE with appropriate molecular weight markers to distinguish between family members

• Gene Expression Analysis Integration:

  • Correlate protein detection with mRNA expression analysis (qRT-PCR with paralog-specific primers)

  • Implement RNA-seq data to determine the expression pattern of all PRICKLE family members in your experimental system

  • Use this information to predict which PRICKLE proteins should be present in your samples

• Subcellular Localization Patterns:

  • Each PRICKLE family member may exhibit distinct subcellular localization patterns

  • Use immunofluorescence with validated antibodies to map localization differences

  • Combine with organelle markers to identify unique compartmentalization patterns

• Knockout Validation Strategy:

  • Generate paralog-specific knockout controls for each PRICKLE family member

  • Test antibody reactivity against these knockout lines to confirm specificity

  • Consider using CRISPR-Cas9 to tag endogenous PRICKLE proteins with different epitope tags for unambiguous identification

What methods can be used to study PRICKLE3's role in cilia growth and positioning?

To investigate PRICKLE3's role in cilia growth and positioning, researchers can implement these methodological approaches:

• High-Resolution Imaging Techniques:

  • Employ super-resolution microscopy (STED, STORM, or SIM) with PRICKLE3 antibody to visualize its precise localization relative to ciliary structures

  • Use live-cell imaging with fluorescently tagged PRICKLE3 to track dynamic changes during ciliogenesis

  • Implement transmission electron microscopy (TEM) with immunogold labeling to determine PRICKLE3's ultrastructural localization

• Co-localization Analysis:

  • Perform double immunostaining with PRICKLE3 antibody and established ciliary markers:

    • Basal body markers (γ-tubulin, centrin)

    • Transition zone proteins (CEP290, NPHP proteins)

    • Axonemal markers (acetylated α-tubulin, IFT proteins)

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

• Functional Perturbation Studies:

  • Generate PRICKLE3 knockdown or knockout models using siRNA or CRISPR-Cas9

  • Assess the impact on:

    • Cilia formation frequency (percentage of ciliated cells)

    • Cilia length (using 3D reconstruction from Z-stack images)

    • Basal body docking and orientation (using polarity markers)

    • Ciliary protein trafficking (using IFT particle visualization)

• Rescue Experiments:

  • Re-express wild-type or mutant PRICKLE3 in knockout models to determine structure-function relationships

  • Create domain deletion constructs to identify which regions are critical for ciliary functions

  • Develop inducible expression systems to study temporal requirements of PRICKLE3 during ciliogenesis

• Ciliary Function Assays:

  • Analyze ciliary signaling pathways (Hedgehog, Wnt) in PRICKLE3-depleted cells

  • Examine calcium influx through ciliary channels using calcium-sensitive fluorescent indicators

  • Assess ciliary motility in multiciliated cells after PRICKLE3 manipulation

• In vivo Models:

  • Study ciliopathy-related phenotypes in animal models with PRICKLE3 mutations

  • Examine organ systems dependent on proper cilia function (brain, kidney, heart) for developmental abnormalities

  • Use tissue-specific conditional knockout approaches to distinguish primary from secondary effects

What are common technical challenges when using PRICKLE3 antibody and how can they be overcome?

Researchers commonly encounter several technical challenges when working with PRICKLE3 antibody. Here are evidence-based solutions for overcoming these issues:

• High Background in Western Blots:

  • Challenge: Non-specific binding producing background signals

  • Solutions:

    • Increase blocking time and concentration (try 5% BSA instead of milk)

    • Optimize antibody dilution (test dilutions from 1:1000 to 1:5000)

    • Increase washing duration and frequency (5 washes for 5-10 minutes each)

    • Add 0.1-0.5% Tween-20 to washing buffer to reduce hydrophobic interactions

    • Consider using more stringent blocking agents (casein or commercial blockers)

• Weak or Absent Signal in IHC:

  • Challenge: Insufficient epitope exposure or antibody penetration

  • Solutions:

    • Optimize antigen retrieval conditions (test different pH buffers and heating times)

    • Decrease antibody dilution (start at 1:50 rather than 1:100)

    • Extend primary antibody incubation time (overnight at 4°C)

    • Use signal amplification systems (polymer-based detection or tyramide signal amplification)

    • Test different fixation protocols if working with fresh tissue samples

• Multiple Bands in Western Blot:

  • Challenge: Detection of splice variants, degradation products, or non-specific proteins

  • Solutions:

    • Ensure complete denaturation of samples (boil longer in sample buffer)

    • Use freshly prepared lysates with protease inhibitors

    • Increase gel percentage for better resolution around target molecular weight

    • Run PRICKLE3 knockdown or knockout controls in parallel

    • Perform peptide competition assays to identify specific bands

• Variable Results Between Experiments:

  • Challenge: Inconsistent antibody performance across experiments

  • Solutions:

    • Aliquot antibody upon first use to avoid freeze-thaw cycles

    • Standardize lysate preparation and protein quantification methods

    • Include validated positive controls in each experiment

    • Maintain consistent antibody incubation times and temperatures

    • Consider lot-to-lot variations and request certificate of analysis when ordering

• Cross-reactivity with Related Proteins:

  • Challenge: PRICKLE3 antibody detecting other PRICKLE family members

  • Solutions:

    • Verify antibody specificity using overexpression and knockout controls

    • Run parallel blots with antibodies against other PRICKLE family members

    • Use cell lines with known expression profiles of PRICKLE family members

    • Consider using peptide pre-absorption controls

How should researchers interpret PRICKLE3 expression patterns in different subcellular compartments?

Interpreting PRICKLE3 subcellular localization patterns requires careful analysis within the context of its known functions. Here is a methodological framework for interpretation:

• Membrane/Cell Junction Localization:

  • Observation: PRICKLE3 at cell-cell junctions or plasma membrane

  • Interpretation: Consistent with its role in the planar cell polarity pathway, where PRICKLE3 functions in the core module at adherens junctions

  • Validation Approach: Co-stain with junction markers (E-cadherin, β-catenin) and other PCP components to confirm functional complexes

  • Functional Significance: May indicate active PCP signaling and establishment of asymmetric protein distribution

• Cytoplasmic Distribution:

  • Observation: Diffuse or punctate cytoplasmic staining

  • Interpretation: Could represent inactive pools, transport intermediates, or interaction with cytoskeletal elements

  • Validation Approach: Co-localization studies with cytoskeletal markers (actin, microtubules) and vesicular transport markers

  • Functional Significance: May indicate regulation of PRICKLE3 availability for membrane recruitment or turnover

• Basal Body/Centrosomal Localization:

  • Observation: PRICKLE3 at centrosome or basal body of cilia

  • Interpretation: Supports known function in basal body organization and cilia growth

  • Validation Approach: Co-stain with centrosomal markers (γ-tubulin, centrin) and ciliary markers (acetylated tubulin)

  • Functional Significance: Suggests active involvement in ciliogenesis or centrosome positioning

• Nuclear Localization:

  • Observation: Nuclear or perinuclear staining

  • Interpretation: Potential novel function in transcriptional regulation or nuclear processes

  • Validation Approach: Nuclear fractionation followed by Western blot; co-staining with nuclear envelope markers

  • Functional Significance: May indicate previously uncharacterized roles in gene expression regulation

• Dynamic Redistribution:

  • Observation: Changes in localization pattern upon stimulation or during development

  • Interpretation: Regulatory mechanisms controlling PRICKLE3 function

  • Validation Approach: Time-course experiments with various stimuli or developmental stages

  • Functional Significance: May reveal activation/inactivation mechanisms or developmental switches in function

• Cell Cycle-Dependent Patterns:

  • Observation: Localization changes throughout cell cycle phases

  • Interpretation: Potential role in cell division or cell cycle regulation

  • Validation Approach: Co-staining with cell cycle markers; synchronized cell populations

  • Functional Significance: Could indicate broader functions beyond established PCP and ciliary roles

How can researchers distinguish between specific and non-specific signals when using PRICKLE3 antibody?

Distinguishing between specific and non-specific signals when using PRICKLE3 antibody requires implementing rigorous validation controls and analytical approaches:

• Genetic Validation Controls:

  • Gold Standard: Compare signal between wild-type and PRICKLE3 knockout/knockdown samples

    • Specific signals will be absent or significantly reduced in knockout samples

    • Non-specific signals will persist regardless of PRICKLE3 expression

  • Implementation: Generate CRISPR knockout cell lines or use siRNA knockdown with >90% efficiency

  • Analysis Method: Quantify signal intensity reduction and determine threshold for specificity

• Peptide Competition Assay:

  • Methodology: Pre-incubate PRICKLE3 antibody with excess immunizing peptide (9-24AA)

  • Expected Result: Specific signals should be blocked or significantly reduced

  • Analysis: Compare signal intensity between competed and non-competed samples

  • Quantification: Calculate percent signal reduction; specific signals typically show >80% reduction

• Signal Characteristics Assessment:

  • Western Blot:

    • Specific signals appear at predicted molecular weight (69 kDa for PRICKLE3)

    • Band intensity correlates with known expression levels across different cell types

    • Signal increases proportionally with protein loading amount

  • IHC/IF:

    • Specific signals show subcellular localization consistent with known biology

    • Staining pattern is reproducible across multiple samples and experimental replicates

    • Signal intensity correlates with transcript levels across tissues

• Secondary Antibody Controls:

  • Implementation: Omit primary antibody while maintaining all other steps

  • Interpretation: Any signal in secondary-only controls represents non-specific binding

  • Application: Adjust blocking conditions or secondary antibody dilution to minimize background

• Isotype Controls:

  • Methodology: Use non-specific IgG from the same host species at matching concentration

  • Analysis: Compare isotype control signal to PRICKLE3 antibody signal

  • Interpretation: True specific signal should exceed isotype control signal by ≥3-fold

• Orthogonal Detection Methods:

  • Approach: Verify PRICKLE3 detection using independent methods

  • Examples:

    • Correlate protein detection with mRNA levels (qRT-PCR)

    • Use multiple antibodies targeting different epitopes

    • Employ tagged PRICKLE3 constructs with detection via tag-specific antibodies

  • Analysis: Concordance between methods increases confidence in specificity

What are the current methodological limitations in PRICKLE3 research and emerging techniques to address them?

Current methodological limitations in PRICKLE3 research present significant challenges, but emerging techniques offer promising solutions:

• Limitation: Limited temporal resolution of protein dynamics

  • Current challenge: Traditional immunostaining provides static snapshots of PRICKLE3 localization

  • Emerging solutions:

    • Live-cell imaging with fluorescently tagged PRICKLE3 constructs

    • Optogenetic tools to control PRICKLE3 activity with light-induced precision

    • FRAP (Fluorescence Recovery After Photobleaching) to measure protein turnover rates

    • Single-molecule tracking to monitor PRICKLE3 movement within cellular compartments

• Limitation: Difficulty distinguishing PRICKLE family members

  • Current challenge: High sequence homology between PRICKLE proteins complicates specific detection

  • Emerging solutions:

    • CRISPR-based endogenous tagging of individual PRICKLE genes

    • Paralog-specific nanobodies with minimal epitope requirements

    • Highly multiplexed imaging using DNA-PAINT or sequential immunofluorescence

    • Proteomics approaches using targeted mass spectrometry with paralog-specific peptides

• Limitation: Unclear protein-protein interaction dynamics

  • Current challenge: Static detection methods miss transient or conditional interactions

  • Emerging solutions:

    • Proximity labeling techniques (BioID, APEX) to capture interaction networks

    • Split-protein complementation assays for real-time interaction monitoring

    • Single-molecule co-tracking to visualize protein complex formation

    • Förster Resonance Energy Transfer (FRET) sensors to detect conformational changes

• Limitation: Tissue-specific functions remain poorly characterized

  • Current challenge: Most studies focus on cell lines rather than complex tissues

  • Emerging solutions:

    • Spatial transcriptomics combined with protein detection

    • Organoid models to recapitulate tissue complexity

    • Tissue-specific conditional knockouts in model organisms

    • Patient-derived samples with known PRICKLE3 mutations

• Limitation: Weak antibody performance in certain applications

  • Current challenge: Current antibodies may have application-specific limitations

  • Emerging solutions:

    • Development of recombinant antibodies with defined binding properties

    • Nanobodies with improved tissue penetration

    • Aptamer-based detection alternatives

    • Machine learning approaches to predict optimal antibody conditions

• Limitation: Difficulty studying low abundance protein

  • Current challenge: PRICKLE3 may be expressed at low levels in certain tissues

  • Emerging solutions:

    • Super-resolution microscopy to detect sparse protein molecules

    • Signal amplification methods (tyramide signal amplification, rolling circle amplification)

    • Single-cell proteomics approaches

    • Targeted protein degradation tools (e.g., dTAG system) to verify signal specificity

How does PRICKLE3 antibody performance compare with antibodies targeting other planar cell polarity proteins?

When evaluating PRICKLE3 antibody performance against antibodies targeting other planar cell polarity (PCP) pathway proteins, researchers should consider these comparative metrics and methodological considerations:

Performance factors to consider when selecting between PCP protein antibodies:

• Epitope Accessibility Comparison:

  • PRICKLE3 antibody (PACO61959) targets an accessible N-terminal region (9-24AA)

  • Transmembrane PCP proteins (CELSR, FRIZZLED) often require more aggressive antigen retrieval

  • Cytoplasmic proteins (DISHEVELLED) generally offer better epitope accessibility

• Signal-to-Noise Ratio Analysis:

  • PRICKLE3 antibody demonstrates strong signal-to-noise ratio in Western blot applications

  • VANGL antibodies typically show comparable performance in immunofluorescence

  • DISHEVELLED antibodies often require careful optimization to avoid background

• Application-Specific Performance:

  • For co-immunoprecipitation: DISHEVELLED antibodies often outperform PRICKLE3 antibodies

  • For tissue immunohistochemistry: PRICKLE3 and CELSR antibodies show strong performance

  • For live-cell applications: FRIZZLED antibodies targeting extracellular domains are advantageous

• Methodological Adaptations Required:

  • PRICKLE3: Standard protocols typically sufficient

  • CELSR: Often requires specialized membrane protein extraction methods

  • FRIZZLED: May benefit from non-permeabilizing conditions for surface detection

  • DISHEVELLED: Phosphatase inhibitors critical due to multiple phosphorylation sites

How can researchers integrate PRICKLE3 antibody studies with genomic and transcriptomic data?

Integrating PRICKLE3 antibody-based protein studies with genomic and transcriptomic data provides comprehensive insights into regulatory mechanisms. Here's a methodological framework for this integration:

• Correlation Analysis Between Protein and mRNA Levels:

  • Methodology:

    • Quantify PRICKLE3 protein levels via Western blot with PRICKLE3 antibody across multiple cell lines/tissues

    • Measure PRICKLE3 mRNA levels in the same samples using qRT-PCR or RNA-seq

    • Calculate Pearson or Spearman correlation coefficients between protein and mRNA datasets

  • Interpretation:

    • Strong correlation suggests transcriptional regulation dominates

    • Poor correlation indicates post-transcriptional regulatory mechanisms

    • Tissue-specific variations may reveal context-dependent regulation

• Genetic Variant Impact Assessment:

  • Methodology:

    • Identify genomic variants in PRICKLE3 from sequencing data or databases

    • Generate cell models expressing variant forms of PRICKLE3

    • Compare protein expression, stability, and localization using PRICKLE3 antibody

  • Applications:

    • Evaluate how coding variants affect protein function

    • Determine if regulatory variants alter expression levels

    • Connect genotype to phenotype through protein-level changes

• Epigenetic Regulation Studies:

  • Methodology:

    • Correlate PRICKLE3 protein levels (detected by antibody) with epigenetic marks at the PRICKLE3 locus

    • Treat cells with epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

    • Monitor changes in PRICKLE3 protein expression using validated antibody

  • Analysis:

    • Integrate ChIP-seq data for histone modifications with protein expression data

    • Map enhancer elements that regulate PRICKLE3 expression

• Alternative Splicing Assessment:

  • Methodology:

    • Identify PRICKLE3 splice variants from RNA-seq data

    • Design isoform-specific primers for qRT-PCR validation

    • Compare protein sizes detected by PRICKLE3 antibody with predicted splice variant sizes

  • Technical Considerations:

    • Verify antibody epitope presence in all splice variants

    • Use high-resolution gel systems to distinguish closely sized isoforms

• Multi-omics Integration Approaches:

  • Methodology:

    • Implement computational methods to integrate protein, transcript, and genomic datasets

    • Apply machine learning to identify patterns across data types

    • Create network models incorporating PRICKLE3 protein interactions and gene regulatory networks

  • Visualization:

    • Develop multi-layer visualizations showing protein-level data alongside genomic/transcriptomic data

    • Use circos plots or heatmaps to represent integrated datasets

What specialized techniques can be used to study PRICKLE3 in rare or difficult-to-access tissues?

Studying PRICKLE3 in rare or difficult-to-access tissues requires specialized methodological approaches that maximize information from limited samples:

• Laser Capture Microdissection (LCM) Combined with Sensitive Detection:

  • Methodology:

    • Prepare thin tissue sections (5-10 μm) on specialized LCM slides

    • Identify regions of interest microscopically

    • Precisely capture specific cell populations using laser cutting

    • Extract protein from captured material using micro-extraction techniques

    • Detect PRICKLE3 using highly sensitive Western blot protocols with PRICKLE3 antibody

  • Sensitivity Enhancements:

    • Use microfluidic Western blot platforms requiring nanogram amounts of protein

    • Implement tyramide signal amplification for immunohistochemistry

    • Apply capillary electrophoresis-based protein analysis for minimal sample requirements

• Single-Cell Protein Analysis:

  • Methodology:

    • Dissociate tissue into single-cell suspensions

    • Perform flow cytometry with PRICKLE3 antibody for quantification across cell populations

    • Implement CyTOF (mass cytometry) for multiplexed protein detection

    • Use imaging flow cytometry to correlate PRICKLE3 expression with morphological features

  • Advanced Applications:

    • Single-cell Western blot to quantify PRICKLE3 in individual cells

    • Proximity extension assay (PEA) for protein detection from minimal sample volumes

• Ex Vivo Tissue Culture Systems:

  • Methodology:

    • Establish organotypic slice cultures from rare tissues

    • Maintain native tissue architecture while allowing experimental manipulation

    • Apply PRICKLE3 antibody for live tissue imaging or fixed tissue analysis

  • Applications:

    • Test drug responses on PRICKLE3 expression or localization

    • Examine dynamic PRICKLE3 behavior in intact tissue microenvironment

• Proximity Labeling in Intact Tissues:

  • Methodology:

    • Express PRICKLE3 fused to biotin ligase (BioID or TurboID) in model organisms

    • Allow in vivo biotinylation of proximity partners

    • Harvest tissue and detect biotinylated proteins alongside PRICKLE3

  • Advantages:

    • Maps tissue-specific PRICKLE3 interaction networks

    • Captures transient interactions within native tissue context

• Spatial Transcriptomics with Protein Validation:

  • Methodology:

    • Perform spatial transcriptomics to map PRICKLE3 mRNA distribution

    • Validate protein expression in serial sections using PRICKLE3 antibody

    • Implement multiplexed immunofluorescence for co-expression analysis

  • Integration:

    • Correlate spatial patterns of transcript and protein

    • Identify discrepancies suggesting post-transcriptional regulation

• Expansion Microscopy:

  • Methodology:

    • Physically expand tissue samples using polymer embedding

    • Apply PRICKLE3 antibody for immunostaining

    • Image with standard microscopes to achieve super-resolution-like results

  • Advantages:

    • Improves spatial resolution without specialized microscopy

    • Enhances detection of low-abundance proteins in complex tissues

How can PRICKLE3 antibody be used in high-throughput screening approaches?

PRICKLE3 antibody can be effectively integrated into high-throughput screening platforms through these methodological implementations:

• Automated Immunofluorescence-Based Screens:

  • Methodology:

    • Culture cells in 96 or 384-well plate formats

    • Apply treatments (small molecules, siRNA libraries, CRISPR libraries)

    • Perform automated fixation and immunostaining with PRICKLE3 antibody

    • Implement high-content imaging using automated microscopy

  • Quantitative Readouts:

    • PRICKLE3 expression levels (total fluorescence intensity)

    • Subcellular localization patterns (nuclear/cytoplasmic ratio, membrane association)

    • Co-localization with other cellular components

    • Morphological changes correlated with PRICKLE3 alterations

  • Analysis Pipeline:

    • Machine learning algorithms for pattern recognition

    • Automated image segmentation and feature extraction

    • Statistical methods to identify significant hits

• Reverse Phase Protein Array (RPPA):

  • Methodology:

    • Spot lysates from treated cells onto nitrocellulose-coated slides

    • Probe arrays with validated PRICKLE3 antibody

    • Detect signal using fluorescent or chemiluminescent methods

    • Analyze signal intensity across treatment conditions

  • Advantages:

    • Extremely low sample requirement (<1μl per spot)

    • Ability to screen hundreds to thousands of conditions simultaneously

    • Quantitative assessment of protein expression changes

• Cell-Based ELISA Platforms:

  • Methodology:

    • Grow cells directly in microplate wells

    • Apply experimental treatments

    • Fix cells and perform in-cell ELISA using PRICKLE3 antibody

    • Quantify signal using plate reader

  • Optimization Requirements:

    • Antibody dilution (recommended 1:2000-1:10000 for ELISA)

    • Blocking conditions to minimize background

    • Signal normalization using housekeeping proteins or cell number

• Flow Cytometry-Based Screens:

  • Methodology:

    • Process cells in 96-well format

    • Permeabilize for intracellular PRICKLE3 detection

    • Stain with PRICKLE3 antibody and fluorescently labeled secondary antibody

    • Analyze using high-throughput flow cytometry systems

  • Applications:

    • Multi-parametric analysis correlating PRICKLE3 with other cellular markers

    • Cell cycle-dependent changes in PRICKLE3 expression

    • Identification of compound effects on specific cell subpopulations

• Bead-Based Multiplex Assays:

  • Methodology:

    • Conjugate PRICKLE3 antibody to uniquely coded microbeads

    • Incubate with cell lysates from experimental conditions

    • Add detection antibody and fluorescent reporter

    • Analyze using multiplex readers (e.g., Luminex platform)

  • Advantages:

    • Simultaneous quantification of PRICKLE3 alongside other proteins

    • Reduced sample requirements compared to traditional Western blot

    • High reproducibility and throughput

• Automated Western Blot Systems:

  • Methodology:

    • Utilize capillary-based automated Western platforms

    • Process multiple samples simultaneously

    • Detect PRICKLE3 using validated antibody at 1:1000-1:5000 dilution

    • Quantify results using integrated analysis software

  • Benefits:

    • Reduced antibody consumption

    • Higher reproducibility than traditional Western blots

    • Faster throughput for screening applications

Future directions for PRICKLE3 research and antibody applications

The future of PRICKLE3 research holds promising avenues for exploration, with antibody-based methodologies playing a central role in advancing our understanding of this protein's functions. Several key directions are emerging:

• Integration with Advanced Imaging Technologies: The application of PRICKLE3 antibody in super-resolution microscopy and expansion microscopy promises to reveal unprecedented details about its subcellular localization and dynamic behavior. These approaches will likely uncover nuanced patterns of PRICKLE3 distribution that have remained inaccessible with conventional microscopy techniques. Similarly, the development of live-cell imaging approaches using tagged PRICKLE3 validated against antibody staining will enable real-time monitoring of protein dynamics during developmental processes and in response to cellular stimuli.

• Systems Biology Approaches: Future research will likely focus on placing PRICKLE3 within larger protein networks through comprehensive interactome mapping. The combination of PRICKLE3 antibody-based co-immunoprecipitation with mass spectrometry will facilitate the identification of context-specific protein interactions. These efforts will be complemented by computational approaches integrating proteomic data with transcriptomic and genomic information, providing a holistic view of PRICKLE3 regulation and function across cellular contexts.

• Therapeutic Target Exploration: As our understanding of PRICKLE3's role in developmental processes and potential disease contexts expands, there will be increasing interest in exploring it as a therapeutic target. PRICKLE3 antibody will be instrumental in validating target engagement in drug discovery pipelines and for characterizing the effects of small molecule modulators on PRICKLE3 expression, localization, and function. This may lead to novel therapeutic strategies for developmental disorders or cancers where planar cell polarity pathways are dysregulated.

• Disease-Specific Applications: Future research will likely focus on characterizing PRICKLE3 expression and localization in human pathological samples, particularly in conditions involving ciliopathies, epithelial cancers, and developmental disorders. The development of more sensitive detection methods using PRICKLE3 antibody will enable analysis in limited clinical samples, potentially revealing biomarker applications or new disease mechanisms.

• Technical Innovations: The development of next-generation antibody formats, such as recombinant monoclonal antibodies against PRICKLE3 with defined epitopes, will enhance reproducibility and specificity. Similarly, the creation of nanobodies or aptamers targeting PRICKLE3 may overcome current limitations in tissue penetration or fixation sensitivity. These innovations will expand the toolkit available for PRICKLE3 research and potentially open new experimental avenues previously limited by antibody technology constraints .