PRICKLE2 Antibody

What epitopes should researchers target when selecting a PRICKLE2 antibody?

When selecting a PRICKLE2 antibody, researchers should consider antibodies targeting either the LIM domain (amino acids 127-323) or the C-terminal region containing the C2 domain (amino acids 585-844). The C-terminal region appears to be the strongest binding site for interactions with other proteins such as AnkG . Custom-made antibodies against these regions have successfully identified PRICKLE2 at the axon initial segment (AIS) of neurons in multiple brain regions . When validating antibody specificity, it's essential to confirm that the antibody recognizes only PRICKLE2 and not the closely related PRICKLE1 protein through appropriate controls .

What are the optimal fixation and permeabilization conditions for PRICKLE2 immunostaining?

For successful PRICKLE2 immunodetection in neuronal cultures and brain sections, standard paraformaldehyde (PFA) fixation (4% PFA) is generally effective. The search results don't specify exact permeabilization conditions, but standard protocols using either 0.1-0.3% Triton X-100 or 0.1% saponin would be appropriate for accessing intracellular PRICKLE2. When co-staining for PRICKLE2 with other AIS markers like AnkG, these fixation conditions allow for excellent colocalization detection as demonstrated in numerous developmental time points (P6, P14, P21) and in cultured neurons at various days in vitro (DIV1, DIV4, and beyond) .

In which neural tissues and subcellular compartments is PRICKLE2 most reliably detected?

PRICKLE2 is prominently detected in several regions of the brain, with notable expression in the cerebral cortex and hippocampus . At the subcellular level, PRICKLE2 shows strong enrichment at the axon initial segment (AIS) where it colocalizes with AnkG, the AIS master organizer . While PRICKLE2 is also present in the postsynaptic density (PSD) as confirmed by colocalization with PSD95, significantly higher immunofluorescence levels are detected at the AIS . During early neuronal development, PRICKLE2 can be found in the soma and in many neurites of unpolarized neurons, but becomes increasingly concentrated in the developing axon and ultimately at the AIS as neurons mature .

How can researchers distinguish between specific and non-specific binding of PRICKLE2 antibodies?

Researchers should implement multiple validation strategies to confirm PRICKLE2 antibody specificity. First, antibodies should be tested against samples where PRICKLE2 has been knocked down using validated shRNA constructs (e.g., shPrickle2A: 5'-GCTGGAGAGAAGTTGCGAA-3' or shPrickle2B) . The search results indicate that effective knockdown should result in significant reduction of immunofluorescence signal (60-75% decrease) . Second, researchers should ensure the antibody does not cross-react with PRICKLE1 by testing on samples expressing only one of these proteins . Finally, immunoprecipitation followed by Western blot should detect the expected 100-kDa band for full-length PRICKLE2, though an additional uncharacterized 70-kDa band has also been observed in rat cortical lysates .

What are the methodological approaches for studying PRICKLE2-AnkG interactions?

Several complementary techniques have proven effective for investigating PRICKLE2-AnkG interactions. Coimmunoprecipitation (coIP) using PRICKLE2 antibodies can pull down AnkG from brain lysates (validated in P21 rat cortical lysates) . For in vitro validation, GST pulldown assays using GST-tagged PRICKLE2 domains can identify specific interaction regions with AnkG . Researchers can also employ cellular assays in COS-7 cells by co-expressing Flag-tagged PRICKLE2 with GFP-tagged AnkG isoforms (especially AnkG480-GFP) to visualize their colocalization and functional interactions . For quantitative assessment of these interactions, it's advisable to analyze multiple microscopic fields and calculate the percentage of cells exhibiting colocalization patterns or specific phenotypes (such as microtubule bundling) under different experimental conditions .

How can researchers effectively quantify PRICKLE2 levels at the AIS in knockdown experiments?

Quantification of PRICKLE2 levels at the AIS following knockdown requires careful immunofluorescence analysis. Based on published methodologies, researchers should:

• Transfect neurons with shRNA constructs targeting PRICKLE2 (e.g., shPrickle2A or shPrickle2B) alongside a control shRNA .

• Perform double immunostaining for PRICKLE2 and AIS markers like AnkG.

• Acquire confocal images using identical settings for all conditions.

• Measure fluorescence intensity along the AIS using line scan analysis.

• Compare relative immunofluorescence levels between control and knockdown neurons.

In published studies, effective knockdown resulted in a 62-75% reduction in PRICKLE2 immunofluorescence at the AIS, with corresponding 57-71% reductions in AnkG levels . Researchers should classify neurons into phenotypic categories (single-AIS/axon, multiple-AIS/axon, or no-AIS phenotypes) based on the pattern of AnkG and PRICKLE2 staining, as these phenotypes correlate with the degree of PRICKLE2 knockdown .

What experimental approaches can reveal the functional consequences of PRICKLE2 depletion on neuronal polarity?

To investigate how PRICKLE2 affects neuronal polarity, researchers should implement a comprehensive experimental strategy:

• Transfect primary hippocampal neurons at the time of plating with shRNA constructs targeting PRICKLE2 .

• Allow neurons to develop in vitro for an appropriate period (10-14 days).

• Immunostain for axonal markers (AnkG) and dendritic markers.

• Classify neurons based on structural phenotypes: single-AIS/axon (normal polarity), multiple-AIS/axon (disrupted polarity), or no-AIS (severe disruption) .

• Quantify the percentage of neurons displaying each phenotype.

Published data shows that approximately 64% of PRICKLE2-depleted neurons develop a multiple-AIS/axon phenotype, indicating altered neuronal polarity with more than three neurites becoming immunoreactive for AnkG/PRICKLE2 . For rescue experiments, researchers should co-express an RNAi-resistant PRICKLE2 mutant (PK2rs) containing mutations in the shRNA target sequence . This approach can confirm the specificity of the observed polarity defects to PRICKLE2 depletion.

How can PRICKLE2 antibodies be used to investigate the molecular mechanisms of AIS assembly?

PRICKLE2 antibodies can be instrumental in studying AIS assembly through several approaches:

• Developmental studies: Immunolabeling neurons at various developmental stages (from DIV1 to DIV22) can reveal the temporal dynamics of PRICKLE2 localization during AIS formation . At DIV1 in stage 2 (unpolarized) neurons, PRICKLE2 and AnkG accumulate in many neurites, while at the onset of axonal specification (stage 2/3), they form aggregates in a fragmented profile along the nascent axon .

• Co-localization analysis: Double-immunolabeling for PRICKLE2 and AnkG, followed by linescan analysis, demonstrates that these proteins fully colocalize in a cohesive pattern as the AIS matures and shortens from DIV4 onward .

• Cellular assays: Transfection of COS-7 cells with PRICKLE2 and AnkG constructs provides a system to study their interactions with the cytoskeleton. Co-expression results in colocalization along thick stretches immunoreactive for tyrosinated tubulin, resembling bundled microtubules . This assay reveals that PRICKLE2 enhances AnkG's ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation .

What controls should be included when using PRICKLE2 antibodies in studies of autism spectrum disorder and epilepsy models?

When studying PRICKLE2 in disease models, several critical controls should be incorporated:

• Genetic verification: Confirm genotypes of animal models, especially if using PRICKLE2-knockout mice which show increased sensitivity to seizures and autism-related behaviors .

• Antibody validation: Include samples from PRICKLE2-knockdown or knockout tissues to confirm antibody specificity in the disease model context .

• Developmental comparisons: Examine PRICKLE2 expression at multiple developmental time points (e.g., P6, P14, P21) as its distribution may change during development .

• Brain region specificity: Compare PRICKLE2 levels across multiple brain regions, as regional differences may correlate with disease manifestations .

• Protein interaction controls: When studying PRICKLE2's interactions with disease-relevant partners like AnkG or Igsf9b, include appropriate negative controls (e.g., non-interacting protein domains) in coimmunoprecipitation or pulldown assays .

• Functional readouts: Correlate PRICKLE2 antibody staining patterns with functional measurements such as neuronal excitability or network activity, which are affected by PRICKLE2 depletion .

What are effective strategies for simultaneously detecting PRICKLE2 and its binding partners in immunofluorescence studies?

Successfully co-detecting PRICKLE2 and its binding partners requires careful antibody selection and optimization:

• Antibody species selection: Choose primary antibodies raised in different host species (e.g., rabbit anti-PRICKLE2 and mouse anti-AnkG) to enable simultaneous detection .

• Sequential staining: If antibodies are from the same species, consider sequential staining protocols with an intermediate blocking step using excess unconjugated secondary antibody.

• Signal amplification: For weaker signals, implement tyramide signal amplification or use highly cross-adsorbed secondary antibodies to minimize cross-reactivity.

• Confocal microscopy settings: Carefully adjust laser power and detector gain to capture both strong AIS signals (where PRICKLE2 is enriched) and weaker signals in other compartments (like the PSD) .

• Controls for co-localization: Include single-stained samples to confirm absence of bleed-through between channels, especially important when assessing co-localization of PRICKLE2 with AnkG at the AIS or with PSD95 at synapses .

How can researchers design effective PRICKLE2 knockdown experiments to study its role in neuronal function?

Designing effective PRICKLE2 knockdown experiments requires careful consideration of several factors:

For functional studies, researchers should complement morphological analyses with electrophysiological recordings, as PRICKLE2 depletion affects action potential firing and network activity .

What techniques effectively distinguish between PRICKLE2 and the closely related PRICKLE1 protein?

Distinguishing between PRICKLE2 and PRICKLE1 is crucial for antibody-based studies:

• Antibody validation: Test antibodies against samples expressing only PRICKLE1 or PRICKLE2 to confirm specificity. Published studies have validated antibodies that recognize only PRICKLE2 and not PRICKLE1 .

• Western blot analysis: PRICKLE2 should appear at approximately 100 kDa, though an additional uncharacterized 70 kDa band has been observed in rat cortical lysates . Compare this pattern with PRICKLE1's distinct molecular weight.

• Functional assays: While both proteins can promote AnkG-dependent microtubule bundling, PRICKLE1 does so with less efficiency than PRICKLE2 (14.9 ± 3% vs. 36 ± 2.7% for PRICKLE2) . This functional difference can help distinguish their roles.

• Expression pattern analysis: Though related, PRICKLE1 and PRICKLE2 may have distinct expression patterns across brain regions or developmental stages that can be leveraged for identification.

• Genetic approaches: For definitive studies, use specific knockdown or knockout models targeting only one family member, followed by antibody staining to confirm specificity.

How can PRICKLE2 antibodies be used to investigate the link between axon initial segment dysfunction and neurodevelopmental disorders?

PRICKLE2 antibodies offer valuable tools for investigating AIS dysfunction in neurodevelopmental disorders:

• Comparative immunohistochemistry: Compare PRICKLE2 localization and levels at the AIS between control and disease model tissues (autism spectrum disorder or epilepsy models). Published studies show that PRICKLE2 depletion disrupts AIS formation and function, which may contribute to disease pathology .

• Protein interaction networks: Use PRICKLE2 antibodies in proteomic approaches to identify alterations in its interaction partners in disease states. Mass spectrometry has already identified 28 PRICKLE2 interactors in the brain, including Igsf9b, which is associated with psychiatric diseases and seizures .

• AIS cytoarchitecture analysis: Examine how PRICKLE2 levels affect the distribution of other AIS components like voltage-gated Na+ channels in disease models. PRICKLE2 depletion causes defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity .

• Developmental trajectory studies: Track PRICKLE2 localization throughout development in control and disease models to identify critical periods when AIS dysfunction may emerge.

• Therapeutic intervention assessment: Use PRICKLE2 antibodies to evaluate whether therapeutic interventions normalize AIS structure and function in disease models.

What methodological approaches can reveal the relationship between PRICKLE2, Igsf9b, and AnkG in regulating AIS cytoarchitecture?

To investigate the complex relationship between PRICKLE2, Igsf9b, and AnkG, researchers should employ multiple complementary approaches:

• Triple immunofluorescence: Simultaneously detect all three proteins in neuronal cultures or brain sections to analyze their spatial relationships. Published data indicates that Igsf9b localizes along axonal processes in a pattern opposite to AnkG, while PRICKLE2 colocalizes with Igsf9b .

• Sequential knockdown experiments: Compare the effects of PRICKLE2 knockdown, Igsf9b knockdown, and double knockdown on AnkG localization. Igsf9b-knockdown neurons display altered AnkG localization, and PRICKLE2 depletion affects Igsf9b subcellular localization .

• Rescue experiments: Test whether overexpression of one protein can rescue defects caused by knockdown of another. This approach can help establish the hierarchy in this regulatory pathway.

• Domain mapping: Use deletion constructs to identify which domains of each protein are critical for their interactions and functions in AIS organization.

• Live imaging: Track the dynamics of fluorescently tagged versions of these proteins during AIS formation and maintenance to understand their temporal relationships.

These methodological approaches can provide insights into how PRICKLE2 regulates AnkG distribution by controlling the proper localization of Igsf9b, potentially revealing shared pathological mechanisms in ASD and epilepsy .

What are the optimal storage and handling conditions for maintaining PRICKLE2 antibody sensitivity and specificity?

While the search results don't provide specific information about PRICKLE2 antibody storage, standard best practices for antibody handling should be followed:

• Store antibody aliquots at -20°C or -80°C for long-term storage to prevent freeze-thaw cycles.

• For working solutions, store at 4°C with appropriate preservatives (e.g., sodium azide at 0.02%).

• Avoid repeated freeze-thaw cycles by creating single-use aliquots.

• Prior to immunostaining, centrifuge antibody solutions to remove any aggregates.

• Always include positive and negative controls in each experiment to confirm antibody performance.

For PRICKLE2 antibodies specifically, researchers should validate each lot by testing on tissues with known PRICKLE2 expression patterns, such as cerebral cortex and hippocampus sections from P6, P14, or P21 rats, where the antibody should label the AIS and colocalize with AnkG .

How can researchers optimize PRICKLE2 antibody protocols for different experimental applications?

Optimizing PRICKLE2 antibody protocols requires adjustments based on the specific application:

For each application, include appropriate controls and validate results using complementary approaches to ensure robust and reproducible findings.