PRICKLE3 Antibody

What is PRICKLE3 and why is it important in cellular research?

PRICKLE3 (prickle planar cell polarity protein 3) is a 615 amino acid protein with a mass of approximately 68.6 kDa that plays critical roles in several cellular processes. It is a member of the Prickle/espinas/testin protein family and is involved in the planar cell polarity (PCP) pathway, which is essential for the polarization of epithelial cells during morphogenetic processes, including gastrulation and neurulation . Recent research has revealed that PRICKLE3 is directly linked to the biogenesis of ATP synthase by specifically binding to ATP8, making it crucial for mitochondrial function . The protein has subcellular localization in the cell membrane, mitochondria, and cytoplasm, and exhibits wide expression across many tissue types . Studies have also implicated PRICKLE3 mutations in Leber's hereditary optic neuropathy (LHON), highlighting its importance in retinal ganglion cell function . This multifaceted role in cellular processes makes PRICKLE3 a significant target for research across developmental biology, cell biology, and neuroscience.

What types of PRICKLE3 antibodies are available and how do they differ?

PRICKLE3 antibodies are available in several formats to accommodate diverse experimental needs:

Different antibodies target distinct regions of PRICKLE3, including the C-terminal region (aa 500-550) or aa 20-305, allowing researchers to investigate specific structural features . The choice between polyclonal and monoclonal antibodies depends on experimental goals—polyclonals offer higher sensitivity by recognizing multiple epitopes, while monoclonals provide greater specificity for a single epitope . Additionally, some antibodies are conjugated (e.g., FITC-conjugated) for direct detection in applications like flow cytometry .

What are the key applications for PRICKLE3 antibodies in research?

PRICKLE3 antibodies serve multiple experimental applications in molecular and cellular research:

Each application requires specific optimization steps. For instance, Western blot protocols typically detect PRICKLE3 at approximately 69 kDa in human samples from cell lines such as Jurkat and HeLa . When performing IHC, researchers have successfully visualized PRICKLE3 in human colon and heart tissues using appropriate antigen retrieval methods . The selection of application should align with research objectives—whether identifying expression patterns, localizing the protein within cells, or investigating protein interactions.

How should researchers optimize Western blot protocols for PRICKLE3 detection?

Optimizing Western blot protocols for PRICKLE3 detection requires attention to several critical factors:

• Sample preparation:

  • Extract proteins using buffers containing protease inhibitors to prevent degradation

  • For mitochondrial PRICKLE3 detection, consider mitochondrial isolation protocols

  • Include phosphatase inhibitors if investigating potential post-translational modifications

• Gel selection and transfer conditions:

  • Use 8-10% gels for optimal resolution of the 69 kDa PRICKLE3 protein

  • Transfer at lower voltage (30V) overnight for large proteins like PRICKLE3

  • Consider wet transfer systems for more efficient transfer of larger proteins

• Antibody optimization:

  • Initial dilution recommendation: 1:500-1:2000 for primary antibodies

  • Incubate overnight at 4°C to improve signal-to-noise ratio

  • Secondary antibody dilution: 1:50,000-1:100,000 for HRP-conjugated antibodies

• Detection considerations:

  • The expected molecular weight is 69 kDa, consistent with observations across multiple studies

  • For low abundance samples, consider enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • When examining mutant forms (e.g., p.Arg53Trp), be aware that the mutation affects protein stability, potentially reducing band intensity by approximately 55%

For validating antibody specificity, PRICKLE3-knockdown cells have proven effective controls, as demonstrated in studies of ATP synthase biogenesis . When investigating PRICKLE3 interactions with ATP synthase components, consider examining the protein levels of other subunits such as ATP6, ATP8, ATPAF1, and ATP5B, which have shown correlations with PRICKLE3 expression levels .

What are the key considerations for immunofluorescence studies of PRICKLE3?

Immunofluorescence (IF) studies of PRICKLE3 require careful attention to protocol optimization for accurate subcellular localization, particularly given its presence in multiple cellular compartments:

• Fixation and permeabilization optimization:

  • 4% paraformaldehyde (PFA) fixation (10-15 minutes) preserves cellular architecture

  • For mitochondrial PRICKLE3 detection, use 0.2-0.5% Triton X-100 for permeabilization

  • Consider methanol fixation (-20°C, 10 min) as an alternative for some epitopes

• Antibody selection and validation:

  • Antibodies confirmed for IF applications show Pearson coefficients of 0.61 for wild-type and 0.55 for mutant PRICKLE3 when co-stained with mitochondrial markers

  • Primary antibody dilutions typically range from 1:50-1:200

  • Include appropriate controls: primary antibody omission, non-specific IgG controls, and ideally, PRICKLE3-knockout or knockdown cells

• Co-localization studies:

  • Pair PRICKLE3 antibodies with established markers:

    • Mitochondria: UQCRC2, TOMM20, or ATP5F

    • Cell membrane: appropriate membrane markers

    • For PCP pathway studies: co-stain with VANGL1/2, CELSR2, or other PCP components

• Imaging parameters:

  • Z-stack acquisition improves resolution of subcellular localization

  • Deconvolution processing enhances visualization of mitochondrial networks

  • Quantitative co-localization analysis should employ appropriate coefficients (Pearson's, Manders')

When investigating PRICKLE3 mutations (such as p.Arg53Trp), note that research has shown this mutation affects protein stability but not mitochondrial localization . For mitochondrial studies, carboxy terminus HA-tagged wild-type or mutant PRICKLE3 constructs have been successfully used to confirm mitochondrial localization through overlap with mitochondrial protein UQCRC2 .

How can researchers effectively use PRICKLE3 antibodies for immunoprecipitation studies?

Immunoprecipitation (IP) experiments with PRICKLE3 antibodies require specific considerations to successfully capture protein interactions, particularly those involving ATP synthase components and planar cell polarity pathway proteins:

• Lysate preparation optimization:

  • For mitochondrial interactions: isolate intact mitochondria before lysis to enrich PRICKLE3-interacting proteins

  • Use gentle lysis buffers (e.g., 1% digitonin or 0.5-1% NP-40) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to maintain interaction integrity

• IP approach selection:

  • Direct IP: Use validated anti-PRICKLE3 antibodies conjugated to beads

  • Tag-based approach: For recombinant studies, HA-tagged PRICKLE3 has been successfully used with anti-HA antibodies

  • For ATP synthase interactions: ATP Synthase Immunocapture Kit has been effectively combined with HA antibodies in mitochondria overexpressing HA-tagged PRICKLE3

• Interaction verification protocols:

  • Reciprocal IPs confirm bidirectional interactions (e.g., PRICKLE3 and ATP8 reciprocally immunoprecipitate)

  • Controls should include IgG control, input lysate (5-10%), and ideally knockout/knockdown controls

  • For ATP synthase interactions, include negative controls such as ATP5A (α), ATP5B (β), ATP5F (b), ATPAF1, or UQCRC2, which have been shown not to directly precipitate with PRICKLE3

• Washing and elution parameters:

  • Multiple gentle washes (4-5 times) with buffer containing reduced detergent

  • Elution in sample buffer for direct Western blot analysis, or milder conditions for maintaining enzymatic activity in functional studies

Research has demonstrated that PRICKLE3 specifically interacts with ATP synthase through binding to ATP8 (A6L) . Additionally, interactions with VANGL proteins (both non-phosphorylated and phosphorylated forms) have been confirmed through immunoprecipitation, indicating functional roles in the planar cell polarity pathway . When studying VANGL interactions, consider examining phosphorylation status using phospho-specific antibodies, as PRICKLE3 has been shown to differentially affect total VANGL versus phosphorylated VANGL pools .

How can PRICKLE3 antibodies be used to investigate its role in ATP synthase biogenesis?

PRICKLE3 antibodies serve as crucial tools for elucidating the protein's direct involvement in ATP synthase biogenesis and function:

• Investigation of PRICKLE3-ATP synthase interactions:

  • Employ co-immunoprecipitation with PRICKLE3 antibodies followed by Western blotting for ATP synthase subunits, focusing on ATP8 which directly interacts with PRICKLE3

  • Use immunofluorescence co-localization studies with PRICKLE3 antibodies and ATP synthase markers to visualize spatial relationships in intact cells

  • Apply proximity ligation assays (PLA) to confirm direct protein interactions in situ

• Analysis of ATP synthase assembly and stability:

  • Utilize blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by Western blotting with PRICKLE3 antibodies to examine complex V assembly states

  • Compare wild-type cells with those carrying PRICKLE3 mutations (e.g., p.Arg53Trp) or PRICKLE3-knockdown cells to assess effects on complex V assembly

  • Research has shown that PRICKLE3-silenced HeLa cells exhibit approximately 31% decrease in fully assembled complex V, which can be rescued by PRICKLE3 overexpression

• Functional analysis protocols:

  • Use PRICKLE3 antibodies to correlate protein levels with ATP synthesis rates in different cellular conditions

  • Combine with measurements of mitochondrial membrane potential and oxygen consumption

  • Examine expression levels of ATP6, ATP8, ATPAF1, and ATP5B which are significantly decreased in cells carrying the p.Arg53Trp PRICKLE3 mutation

• Disease-relevant research applications:

  • Investigate PRICKLE3 mutations in Leber's hereditary optic neuropathy (LHON) using patient-derived cells

  • Compare cells carrying both ND4 m.11778G>A and PRICKLE3 p.Arg53Trp mutations versus those with single mutations

  • Research indicates that cells with both mutations exhibit greater mitochondrial dysfunction than those with single mutations, suggesting synergistic effects

The development of a pulse-chase assay using cycloheximide (CHX) combined with PRICKLE3 antibody detection has proven effective for studying protein degradation dynamics and the stabilizing effects of PRICKLE3 on its interaction partners .

How can researchers utilize PRICKLE3 antibodies to study its roles in the planar cell polarity pathway?

PRICKLE3 antibodies provide valuable tools for investigating the protein's involvement in the planar cell polarity (PCP) pathway through several methodological approaches:

• Protein complex analysis strategies:

  • Immunoprecipitation with PRICKLE3 antibodies followed by detection of PCP components (particularly VANGL2, CELSR2, and KIF26B)

  • Proximity-based interactome mapping using BioID or APEX approaches with PRICKLE3 as the bait protein

  • Recent proteomic studies using miniTurboID-tagged PRICKLE3 have identified approximately 117 unique interactors and 130 interactors shared with PRICKLE2

• Subcellular localization examination:

  • Co-immunofluorescence staining of PRICKLE3 with PCP pathway components

  • Analysis of protein accumulation at cell-cell contacts during polarization

  • Comparison of PRICKLE3 localization patterns with other PRICKLE family members (PRICKLE1 and PRICKLE2)

  • Research indicates PRICKLE3 has the most distinct interactome compared to PRICKLE1 and PRICKLE2

• Functional interaction assessment:

  • Use PRICKLE3 antibodies in conjunction with VANGL phosphorylation-specific antibodies

  • Analyze how PRICKLE3 affects VANGL stability and phosphorylation through pulse-chase experiments

  • Studies have shown that PRICKLE3 protects VANGL from degradation over time but has no significant effect on the VANGL pool phosphorylated by CK1δ/ε

• Developmental context investigation:

  • Apply PRICKLE3 antibodies in developmental studies examining PCP during morphogenesis

  • Compare expression patterns in epithelial tissues undergoing polarization

  • Analyze the consequences of PRICKLE3 mutations on PCP-dependent developmental processes

For comprehensive analysis, consider using multiple PRICKLE isoform antibodies (PRICKLE1, PRICKLE2, and PRICKLE3) to compare their expression patterns and functional roles. Principal component analysis of interactome data has revealed that PRICKLE3 has the most distinct interaction profile compared to other PRICKLE proteins, with PRICKLE1 and PRICKLE2 sharing more similarities .

What approaches can be used to study PRICKLE3 post-translational modifications using specific antibodies?

Investigating PRICKLE3 post-translational modifications (PTMs) requires specialized antibody-based methodologies to detect and characterize these regulatory events:

• Phosphorylation analysis strategies:

  • Use phospho-specific antibodies when available, or employ general phospho-detection methodologies:

    • Immunoprecipitate PRICKLE3 followed by immunoblotting with anti-phospho-serine/threonine/tyrosine antibodies

    • Phos-tag SDS-PAGE combined with PRICKLE3 antibody detection to resolve phosphorylated species

  • Compare phosphorylation patterns in different cellular contexts (e.g., WNT pathway activation)

  • Consider that CK1 kinases (CK1δ/ε and CK1α) have been identified as significant interactors of PRICKLE proteins

• Ubiquitination detection approaches:

  • Immunoprecipitate PRICKLE3 under denaturing conditions followed by ubiquitin detection

  • His-tagged ubiquitin pulldown assays combined with PRICKLE3 antibody detection

  • Studies have used this approach in HEK T-REx 293 PRICKLE3 TetON cells transfected with His-tagged ubiquitin

• Other PTM investigation methods:

  • SUMOylation: Immunoprecipitate PRICKLE3 followed by SUMO detection

  • Acetylation: Use anti-acetyl-lysine antibodies after PRICKLE3 immunoprecipitation

  • Glycosylation: Treat samples with glycosidases before Western blotting with PRICKLE3 antibodies

• Integration with mass spectrometry:

  • Immunopurify PRICKLE3 using specific antibodies for subsequent MS analysis

  • Enrich for specific PTMs using appropriate methodologies before MS

  • Compare PTM profiles across different cellular conditions or disease states

When investigating PRICKLE3 in relation to the WNT signaling pathway, consider examining phosphorylation of its interaction partners. For instance, VANGL2 contains a cluster of serine/threonine residues (T78, S79, and S82) that are phosphorylation targets for Casein kinase I epsilon and delta (CK1ε/δ), which can be detected using phospho-specific antibodies .

What are common challenges in detecting PRICKLE3 and how can they be overcome?

Researchers frequently encounter several challenges when detecting PRICKLE3 protein, which can be addressed through methodological refinements:

• Low signal intensity issues:

  • Potential causes: Low endogenous expression, antibody sensitivity limitations, protein degradation

  • Solutions:

    • Increase antibody concentration (try 1:500 instead of 1:2000 for Western blot)

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

    • Use enhanced detection systems (high-sensitivity ECL substrates)

    • Consider cell types with higher expression (Jurkat and HeLa cells show detectable levels)

• Multiple band detection:

  • Potential causes: Splice variants (up to 2 isoforms reported) , degradation products, cross-reactivity

  • Solutions:

    • Verify antibody specificity using PRICKLE3-knockdown controls

    • Include protease inhibitors during sample preparation

    • Consider epitope-specific antibodies targeting different regions (N-terminal vs. C-terminal)

• Mitochondrial fraction detection challenges:

  • Potential causes: Insufficient mitochondrial isolation, low enrichment

  • Solutions:

    • Optimize mitochondrial isolation protocols

    • Use proteinase K treatment to differentiate outer membrane-associated vs. inner membrane/matrix PRICKLE3

    • Perform subcellular fractionation with appropriate markers (TOMM20 for outer membrane, ATP5F for inner membrane)

• Inconsistent results across application types:

  • Potential causes: Epitope masking in certain applications, fixation sensitivity

  • Solutions:

    • For IHC/IF: Try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

    • Test multiple antibodies targeting different epitopes

    • For IP: Use gentler lysis conditions to preserve protein-protein interactions

The p.Arg53Trp mutation affects PRICKLE3 stability, reducing protein levels by approximately 55% while not affecting mitochondrial localization . This should be considered when working with samples carrying this mutation, as higher antibody concentrations or longer exposure times may be necessary for detection.

How should researchers validate PRICKLE3 antibody specificity for their experiments?

Rigorous validation of PRICKLE3 antibody specificity is essential for generating reliable research data. A comprehensive validation approach includes:

• Genetic manipulation controls:

  • PRICKLE3 knockdown/knockout: Compare antibody signal between wild-type and PRICKLE3-depleted samples

    • shRNA-based PRICKLE3 knockdown in HeLa cells has been effectively used to validate antibody specificity

    • CRISPR/Cas9-generated PRICKLE3 knockout models provide definitive negative controls

  • Overexpression verification: Detect increased signal in cells overexpressing PRICKLE3

    • HA-tagged or other epitope-tagged PRICKLE3 constructs allow dual verification with tag-specific antibodies

• Application-specific validation methods:

  • Western blot:

    • Verify expected molecular weight (69 kDa)

    • Peptide competition assays to confirm epitope specificity

    • Compare multiple antibodies targeting different epitopes

  • Immunofluorescence/IHC:

    • Appropriate subcellular localization (membrane, mitochondria, cytoplasm)

    • Co-localization with established markers (e.g., UQCRC2 for mitochondria)

    • Primary antibody omission controls

• Cross-species reactivity assessment:

  • Test antibody against PRICKLE3 from multiple species if cross-reactivity is claimed

  • PRICKLE3 orthologs have been reported in mouse, rat, bovine, frog, and chimpanzee species

  • Sequence alignment analysis to predict potential cross-reactivity

• Lot-to-lot consistency evaluation:

  • Compare performance across different antibody lots using standardized samples

  • Maintain reference samples for long-term consistency testing

  • Document optimal working conditions for each lot

For studies involving specific mutations like p.Arg53Trp, include appropriate mutation-carrying samples to assess any potential impacts on antibody binding. Research has demonstrated that this mutation affects protein stability but not epitope recognition by antibodies .

What considerations are important when designing multi-parameter experiments involving PRICKLE3 antibodies?

Multi-parameter experiments involving PRICKLE3 antibodies require careful planning and optimization to generate reliable, integrated datasets:

• Antibody compatibility assessment:

  • Species considerations: When combining multiple primary antibodies, select those raised in different host species

    • Available PRICKLE3 antibodies include rabbit polyclonal, goat polyclonal, and mouse monoclonal options

  • Fluorophore selection: For multiplexed IF, choose non-overlapping fluorescent spectra

  • Application validation: Ensure all antibodies perform reliably in the chosen experimental context

• Co-immunoprecipitation experimental design:

  • Sequential IP strategies: For studying multi-protein complexes involving PRICKLE3

    • First IP with PRICKLE3 antibody followed by second IP targeting interaction partner

    • Particularly useful for studying ATP synthase interactions with confirmation of ATP8 binding

  • Controls: Include appropriate controls for each IP step

  • Buffer optimization: Adjust lysis and washing conditions to maintain specific interactions

• Combined technique approaches:

  • IF-PLA integration: Combine standard IF with proximity ligation assay

    • Use PRICKLE3 antibody for localization plus PLA for specific interaction detection

  • ChIP-Western integration: For transcriptional complex studies

  • Live-cell with fixed-cell correlation: Tag-based live imaging followed by antibody-based fixed analysis

• Quantitative analysis planning:

  • Normalization strategies: Select appropriate housekeeping proteins or total protein normalization

  • Image analysis parameters: Define consistent quantification methods for IF/IHC

  • Statistical approach: Determine appropriate statistical tests based on experimental design

For studies examining PRICKLE3's dual roles in mitochondria and planar cell polarity, consider subcellular fractionation approaches to separately analyze distinct protein pools. Research has shown that cellular fraction experiments can effectively separate mitochondrial PRICKLE3 (co-fractionating with TOMM20 and ATP5F) from cytosolic PRICKLE3 (co-fractionating with tubulin) .

How are PRICKLE3 antibodies being used to understand its role in Leber's hereditary optic neuropathy (LHON)?

PRICKLE3 antibodies have become instrumental in elucidating the protein's involvement in Leber's hereditary optic neuropathy (LHON) through several innovative approaches:

• Genetic-biochemical correlation studies:

  • PRICKLE3 antibodies enable protein level quantification in patient-derived cells carrying the p.Arg53Trp mutation

  • Western blot analysis of mutant lymphoblastoid cell lines has demonstrated an approximately 55% reduction in PRICKLE3 protein levels compared to controls

  • These findings help explain how the p.Arg53Trp mutation acts in synergy with the mitochondrial ND4 11778G>A mutation to cause LHON

• Retinal ganglion cell (RGC) investigation:

  • Immunohistochemistry with PRICKLE3 antibodies in retinal tissues from PRICKLE3-knockout mice

  • Analysis reveals that RGCs (Brn3a-positive) in PRICKLE3−/− mice demonstrate thinner morphology in the RGC layer compared to wild-type mice

  • Patient-derived RGCs carrying both ND4 m.11778G>A and PRICKLE3 p.Arg53Trp mutations show abnormal morphology when examined with appropriate antibodies

• Mitochondrial dysfunction mechanisms:

  • PRICKLE3 antibodies help track ATP synthase assembly and stability in normal versus mutant conditions

  • Combined with functional assays, antibody-based detection has shown that:

    • Cells with both p.Arg53Trp and m.11778G>A mutations display greater mitochondrial dysfunction than those with single mutations

    • ATP production efficiency is reduced in double-mutation carriers

    • Susceptibility to apoptosis is increased in cells carrying both mutations

• Therapeutic target identification:

  • Screening approaches using PRICKLE3 antibodies to identify compounds that might stabilize mutant PRICKLE3

  • Evaluation of potential interventions aimed at rescuing ATP synthase assembly and function in mutation carriers

These research applications highlight how PRICKLE3 antibodies are essential for connecting genetic findings to molecular mechanisms in LHON pathogenesis, potentially leading to new therapeutic approaches for this currently untreatable condition.

What emerging techniques are enhancing the utility of PRICKLE3 antibodies in research?

Novel methodological approaches are expanding the applications and analytical power of PRICKLE3 antibodies:

• Proximity-based interactome mapping:

  • BioID/TurboID approaches: Fusion of PRICKLE3 with biotin ligase to identify proximal proteins

    • MiniTurboID-V5-tagged PRICKLE3 has been successfully used to map interaction networks

    • This approach identified approximately 1100 significant proximal interactors

  • APEX-based methods: Alternative proximity labeling for rapid kinetic studies

  • These techniques complement traditional antibody-based co-immunoprecipitation, providing spatial context to interactions

• Advanced microscopy applications:

  • Super-resolution microscopy: Using PRICKLE3 antibodies for nanoscale localization

    • STED or STORM microscopy to resolve mitochondrial subcompartmentalization

    • Single-molecule localization to track PRICKLE3 dynamics

  • Live-cell compatible immunofluorescence: Using cell-permeable antibody fragments

  • Expansion microscopy: Physical expansion of samples for enhanced resolution of PRICKLE3 distribution

• Integrated multi-omics approaches:

  • IP-mass spectrometry: PRICKLE3 antibody immunoprecipitation coupled with MS analysis

    • Identification of post-translational modifications

    • Discovery of novel interaction partners

  • ChIP-seq integration: For studying potential transcriptional regulatory roles

  • Spatial transcriptomics correlation: Connecting PRICKLE3 protein localization with local transcriptome profiles

• High-throughput screening applications:

  • Antibody-based cellular arrays: Screening for modulators of PRICKLE3 expression/localization

  • CRISPR library screening: Combined with PRICKLE3 antibody readouts to identify genetic interactors

  • Small molecule screening: Using PRICKLE3 antibodies to identify compounds that affect its stability or function