PRICKLE4 Antibody

What is PRICKLE4 and what are its key structural features?

PRICKLE4, also known as over-expressed breast tumor protein (OBTP) or C6orf49, is a member of the LIM family of proteins. The protein possesses two characteristic LIM domains as well as a PET domain and is thought to primarily localize to the nucleus . It weighs approximately 130 kDa and expresses in multiple tissues, with notable presence in the central nervous system and kidneys . The human PRICKLE4 gene is located on chromosome 6 .

The protein shares structural similarities with other PRICKLE family members (PRICKLE1 and PRICKLE2), suggesting functional conservation within this protein family. These structural motifs are critical for protein-protein interactions and likely determine PRICKLE4's biological functions in signaling pathways .

What techniques can PRICKLE4 antibodies be effectively used for?

PRICKLE4 antibodies have been validated for multiple experimental applications:

When performing these techniques, researchers should consider the specific epitope targeted by their antibody. For example, some commercial PRICKLE4 antibodies target synthetic peptides near the amino terminus of human PRICKLE4, which may affect detection in different experimental contexts .

How does PRICKLE4 function within cellular signaling networks?

Current evidence suggests PRICKLE4 likely functions within the noncanonical WNT signaling pathway, similar to its family members PRICKLE1 and PRICKLE2 . This pathway plays critical roles in:

• Regulation of intracellular calcium release

• Establishment and maintenance of planar cell polarity (PCP)

• Coordinated cell movements during development

Additionally, PRICKLE4 has been identified as one of the top three target genes co-expressed with FOXP4-AS1, suggesting its involvement in gene regulatory networks relevant to cancer progression . The protein's nuclear localization further indicates potential roles in transcriptional regulation or chromatin organization, though specific mechanisms require further investigation.

What are the optimal storage and handling conditions for PRICKLE4 antibodies?

To maintain PRICKLE4 antibody functionality and specificity:

• Store at 4°C for short-term use (up to three months)

• Store at -20°C for long-term stability (up to one year)

• Avoid repeated freeze-thaw cycles which significantly reduce antibody performance

• Most commercial PRICKLE4 antibodies are supplied in PBS containing 0.02% sodium azide as a preservative

• Working dilutions should be prepared fresh and used within 24 hours for optimal results

• Antibodies should not be exposed to prolonged high temperatures during shipping or storage

These conditions are critical for maintaining epitope recognition and preventing aggregation or degradation that could compromise experimental outcomes.

How can researchers validate PRICKLE4 antibody specificity?

A multi-faceted approach to antibody validation should include:

• Blocking peptide competition assays: Compare western blot results with and without the specific immunogen peptide. Genuine PRICKLE4 bands should disappear when the blocking peptide is present, as demonstrated in validation studies showing signal reduction in A549 cell lysates when using a blocking peptide .

• Multiple detection methods: Confirm findings across different techniques (WB, ICC, IF) to ensure consistent detection patterns.

• Positive and negative controls: Include tissues/cells known to express PRICKLE4 (e.g., A549 cells) and those with minimal expression.

• siRNA knockdown: Reduce PRICKLE4 expression through siRNA and confirm corresponding reduction in antibody signal.

• Band size verification: Confirm detection at the predicted molecular weight (approximately 38 kDa theoretical size, though observed at ~130 kDa in some studies, suggesting post-translational modifications) .

What protocol optimizations improve PRICKLE4 immunofluorescence detection?

For optimal PRICKLE4 detection in immunofluorescence studies:

• Fixation method: 4% paraformaldehyde provides superior epitope preservation compared to methanol fixation.

• Permeabilization: Use 0.1-0.5% Triton X-100 to ensure antibody access to the nuclear compartment where PRICKLE4 primarily localizes.

• Blocking conditions: Extend blocking time (2-3 hours) with 5% BSA and 2% normal serum from the secondary antibody host species to reduce background.

• Antibody concentration: Titrate between 5-20 μg/mL, as validated studies have shown successful staining at these concentrations .

• Incubation conditions: Overnight incubation at 4°C often yields better signal-to-noise ratio than shorter incubations at room temperature.

• Signal amplification: Consider tyramide signal amplification for low abundance detection while maintaining specificity.

What is the relationship between PRICKLE4 and neurological disorders?

PRICKLE4 has potential implications in neurological conditions through several mechanisms:

• Synaptic regulation: PRICKLE4 may interact with synaptic proteins like synapsin to influence synaptic stability and function .

• Neuronal connectivity: Research suggests PRICKLE4 regulates neuronal connections, potentially contributing to epilepsy pathophysiology through altered network excitability .

• WNT signaling modulation: As a likely component of the noncanonical WNT pathway (similar to other PRICKLE family members), PRICKLE4 may influence neuronal development and plasticity through calcium signaling and cytoskeletal reorganization .

When investigating these relationships, researchers should consider co-localization studies with synaptic markers and functional assays measuring neuronal activity in models with altered PRICKLE4 expression.

How does PRICKLE4 contribute to cancer biology?

PRICKLE4's potential role in oncogenesis is supported by several observations:

• Expression pattern: PRICKLE4 shows expression across multiple malignant tissue differentiations, suggesting a potential role in cancer differentiation processes .

• Alternative name connection: Its alternative name "over-expressed breast tumor protein" (OBTP) indicates historical observation of upregulation in breast cancer contexts .

• Co-expression network: PRICKLE4 was identified as one of the top three genes co-expressed with FOXP4-AS1, a long non-coding RNA associated with poor cancer prognosis . This association places PRICKLE4 within a gene network implicated in critical cancer mechanisms including DNA replication, cell cycle regulation, and DNA conformation changes .

• Pathway involvement: GO and KEGG pathway analyses of PRICKLE4's co-expression network reveal connections to fundamental cancer-related processes including nuclear DNA replication, DNA conformation change, and cell cycle regulation .

Researchers investigating PRICKLE4 in cancer contexts should consider analyzing expression patterns across cancer types, correlating expression with clinical outcomes, and exploring functional consequences of PRICKLE4 modulation in cancer models.

What methodological approaches should be used when studying PRICKLE4 in complex tissue samples?

When investigating PRICKLE4 in heterogeneous tissue samples:

• Multi-antibody validation: Use antibodies targeting different PRICKLE4 epitopes to confirm expression patterns and reduce epitope-specific artifacts.

• Cell type identification: Combine PRICKLE4 immunostaining with cell-type-specific markers to identify which cell populations express the protein within complex tissues.

• Tissue processing considerations:

  • Formalin-fixed paraffin-embedded (FFPE) tissues may require antigen retrieval optimization

  • Fresh-frozen sections often provide superior epitope preservation

  • Consider specialized fixation methods for tissues with high lipid content

• Quantification approaches: Implement digital image analysis with standardized thresholding to objectively quantify PRICKLE4 expression across tissue regions.

• Single-cell techniques: For heterogeneous populations, consider single-cell approaches (flow cytometry, single-cell sequencing) to characterize PRICKLE4 expression at cellular resolution.

How can researchers troubleshoot non-specific binding with PRICKLE4 antibodies?

When encountering non-specific binding:

• Increase blocking stringency: Extend blocking time and use a combination of blocking agents (BSA, normal serum, casein) to reduce non-specific interactions.

• Adjust antibody concentration: Titrate to determine the optimal concentration that maximizes specific signal while minimizing background. Validated studies have used 0.25 μg/mL for Western blot and 5-20 μg/mL for immunocytochemistry .

• Modify washing protocol: Increase wash duration and volume, and consider adding low concentrations of detergent (0.05-0.1% Tween-20) to remove weakly bound antibodies.

• Pre-adsorb antibody: Incubate the antibody with negative control lysates to remove cross-reactive antibodies before application to experimental samples.

• Validate secondary antibody: Test secondary antibody alone to ensure it's not contributing to background signal.

• Consider epitope location: Some commercial PRICKLE4 antibodies target an N-terminal epitope , which may be masked in certain conformational states or protein complexes.

What are the current challenges and limitations in PRICKLE4 research?

Researchers face several challenges when studying PRICKLE4:

• Limited characterization: Compared to other PRICKLE family members, PRICKLE4 has been less extensively studied, resulting in knowledge gaps about its precise functions and interactions.

• Molecular weight discrepancy: While theoretical molecular weight is ~38 kDa, some studies observe a band at ~130 kDa , suggesting significant post-translational modifications that could affect detection and function.

• Structural variability: Like other proteins with multiple domains, PRICKLE4 may adopt various conformations depending on cellular context, affecting epitope accessibility and antibody binding.

• Functional redundancy: Potential functional overlap with other PRICKLE family members may complicate interpretation of phenotypes in knockdown/knockout studies.

• Pathway complexity: The noncanonical WNT pathway, where PRICKLE4 likely functions, involves complex cross-talk with other signaling pathways, making it challenging to isolate PRICKLE4-specific effects.

• Tissue-specific expression: Expression patterns vary across tissues, requiring careful selection of experimental models that appropriately represent the tissue of interest.

How can researchers optimize co-immunoprecipitation protocols for studying PRICKLE4 protein interactions?

For successful co-immunoprecipitation (Co-IP) of PRICKLE4 and its binding partners:

• Lysis buffer optimization: Use buffers that preserve protein-protein interactions (e.g., NP-40 or CHAPS-based buffers) rather than harsh detergents like SDS that disrupt interactions.

• Cross-linking consideration: For transient or weak interactions, implement reversible cross-linking (e.g., DSP or formaldehyde) before lysis.

• Antibody selection: Choose antibodies that target regions of PRICKLE4 unlikely to be involved in protein-protein interactions. N-terminal-directed antibodies may be advantageous as the LIM domains, likely involved in protein interactions, are not directly targeted .

• Binding conditions: Adjust salt and detergent concentrations to balance between maintaining specific interactions and reducing background.

• Controls:

  • Input control (pre-IP lysate)

  • IgG control (non-specific antibody of same isotype)

  • Reciprocal IP (immunoprecipitate with antibodies against suspected binding partners)

• Detection strategy: Consider using highly sensitive detection methods like mass spectrometry to identify novel interaction partners.

What emerging technologies show promise for advancing PRICKLE4 research?

Several cutting-edge approaches could accelerate PRICKLE4 research:

• CRISPR-Cas9 genome editing: Precise modification of PRICKLE4 locus to study domain-specific functions or create reporter systems for live-cell imaging.

• Proximity labeling techniques: BioID or APEX2 fusion proteins to identify the PRICKLE4 interactome in living cells with spatial and temporal resolution.

• Super-resolution microscopy: Technologies like STORM or PALM can reveal PRICKLE4 subcellular localization at nanometer resolution, potentially identifying novel functional compartments.

• Single-cell multi-omics: Combined single-cell transcriptomics and proteomics to understand cell-type-specific PRICKLE4 regulation and function.

• Structural biology approaches: Cryo-EM or X-ray crystallography of PRICKLE4 and its complexes to understand how its LIM and PET domains mediate protein interactions.

• Patient-derived organoids: Culture systems that more faithfully recapitulate complex tissue environments to study PRICKLE4 in physiologically relevant contexts.

What are the implications of PRICKLE4's association with FOXP4-AS1 in cancer research?

The identification of PRICKLE4 as one of the top three genes co-expressed with FOXP4-AS1 opens several research avenues:

• Biomarker potential: Investigating whether PRICKLE4 expression, alone or in combination with FOXP4-AS1, could serve as a prognostic indicator in cancer.

• Regulatory mechanisms: Exploring whether FOXP4-AS1 directly regulates PRICKLE4 expression or if they share upstream regulators, potentially identifying new therapeutic targets.

• Functional synergy: Determining if PRICKLE4 and FOXP4-AS1 functionally cooperate in promoting cancer hallmarks like proliferation, invasion, or therapy resistance.

• Pathway integration: GO and KEGG analyses of the co-expression network implicate processes like DNA replication, cell cycle, and DNA conformation change , suggesting specific cellular mechanisms for investigation.

• Therapeutic implications: Evaluating whether targeting PRICKLE4 could sensitize cancer cells to existing therapies or provide novel treatment approaches.