PAWR Antibody

What is PAWR and why is it a target for antibody development?

PAWR (PRKC, Apoptosis, WT1, Regulator), also known as Par-4 or PAR4, is a proapoptotic protein that selectively induces apoptosis primarily in cancer cells. The protein has become an important research target due to its role in the regulation of programmed cell death pathways. PAWR antibodies are developed to study the expression, localization, and function of this protein in various biological contexts, particularly in cancer research and developmental biology. The protein has also been identified as a novel PITX2-interacting protein that plays a role in ocular cell development, connecting PITX2 to apoptosis pathways in the eye .

What are the common types of PAWR antibodies available for research?

PAWR antibodies are primarily available as polyclonal and monoclonal formats, with rabbit being the most common host species. The main types include:

These antibodies are typically purified through peptide affinity chromatography and are available in unconjugated forms for maximum flexibility in experimental design .

How is the specificity of PAWR antibodies validated?

The specificity of PAWR antibodies is validated through multiple complementary approaches. Initially, antibodies are tested for their ability to detect endogenous levels of total PAWR protein. Validation typically includes Western blot analysis using extracts from various cell lines expressing PAWR at different levels. Additional validation is performed through immunohistochemistry on paraffin-embedded tissues such as human colon cancer tissue. The pattern of staining is compared with known expression profiles of PAWR. For more rigorous validation, some antibodies undergo testing with positive and negative controls, including PAWR-knockout cell lines or tissues and recombinant PAWR protein. Co-immunoprecipitation experiments may also be performed, such as immunoprecipitation with an anti-Xpress antibody followed by immunoblotting with an anti-PAWR antibody to confirm specific protein-protein interactions .

What are the optimal conditions for using PAWR antibodies in Western blotting?

For optimal Western blotting using PAWR antibodies, researchers should consider the following protocol:

• Sample preparation: Prepare cell or tissue lysates in RIPA buffer containing protease inhibitors. For PAWR detection, tissue samples from human, mouse, or rat origins are suitable.

• Protein loading and separation: Load 20-50 μg of total protein per lane on 10-12% SDS-PAGE gels. PAWR has a molecular weight of approximately 38-40 kDa.

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 1-2 hours in Tris-glycine buffer with 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute PAWR antibody (typically 1:500 to 1:2000, depending on the specific antibody) in blocking solution and incubate overnight at 4°C.

• Washing and secondary antibody: Wash membranes 3-5 times with TBST, then incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for most PAWR antibodies) at 1:5000 - 1:10000 dilution for 1 hour at room temperature.

• Detection: After washing, develop using enhanced chemiluminescence (ECL) reagent.

For validation, extracts from various cell lines should be run simultaneously to confirm specificity and expected molecular weight .

How should PAWR antibodies be utilized in immunohistochemistry applications?

For successful immunohistochemistry (IHC) using PAWR antibodies, follow these methodological guidelines:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections of 4-6 μm thickness. Fresh frozen sections may also be used but often yield different staining patterns.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) for 15-20 minutes.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes, followed by protein blocking with 5-10% normal serum.

• Primary antibody: Apply diluted PAWR antibody (typically 1:50 to 1:200) and incubate for 1-2 hours at room temperature or overnight at 4°C.

• Detection system: Use a polymer-based detection system compatible with rabbit primary antibodies, followed by DAB (3,3'-diaminobenzidine) chromogen for visualization.

• Counterstaining: Counterstain with hematoxylin, dehydrate, clear, and mount.

PAWR antibodies have been successfully used to detect expression in human colon cancer tissue, and researchers should include appropriate positive controls (tissues known to express PAWR) and negative controls (primary antibody omission) .

What considerations are important when using PAWR antibodies for co-immunoprecipitation studies?

When performing co-immunoprecipitation (co-IP) with PAWR antibodies to study protein-protein interactions, several key methodological considerations are crucial:

• Lysis conditions: Use mild lysis buffers (e.g., NP-40 or Triton X-100 based) that preserve native protein conformations and interactions. Typically, buffers containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and protease/phosphatase inhibitors are effective.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody selection: For PAWR co-IP, use antibodies specifically validated for immunoprecipitation. Consider using different epitope-targeting antibodies for immunoprecipitation versus immunoblotting to avoid heavy chain interference.

• Control experiments: Include proper controls such as:

  • IgG control (same species as PAWR antibody)

  • Input control (typically 5% of total lysate used for IP)

  • Reverse co-IP (immunoprecipitate with antibody against interacting protein)

• Washing conditions: Wash immunoprecipitates thoroughly (4-5 times) but use buffers with salt concentrations that won't disrupt genuine interactions.

• Detection strategy: For known PAWR interactions (such as with PITX2), immunoprecipitate with anti-Xpress antibody and immunoblot with anti-PAWR antibody, or vice versa, depending on experimental design.

Research has demonstrated successful co-IP of PAWR with interacting proteins such as PITX2, revealing important functional relationships in ocular development and apoptosis regulation .

How can PAWR antibodies be utilized to study its role in cancer-selective apoptosis pathways?

PAWR antibodies enable sophisticated investigation into cancer-selective apoptotic mechanisms through several methodological approaches:

• Differential expression analysis: Compare PAWR expression in matched tumor and normal tissues using immunohistochemistry with PAWR antibodies. Quantify staining intensity and subcellular localization across multiple tissue samples to correlate expression patterns with clinical outcomes.

• Subcellular localization studies: Employ immunofluorescence with PAWR antibodies alongside markers for different cellular compartments to track PAWR translocation between cytoplasm and nucleus during apoptosis induction. This is critical as PAWR's pro-apoptotic function often correlates with its nuclear localization in cancer cells.

• Protein-protein interaction networks: Use PAWR antibodies in proximity ligation assays or co-immunoprecipitation studies to map the dynamic interaction landscape of PAWR with known apoptotic regulators (e.g., PKA, PKCζ, Akt) under various cellular stresses.

• Phosphorylation status monitoring: Utilize phospho-specific PAWR antibodies to track post-translational modifications that regulate PAWR's pro-apoptotic activity, particularly in response to therapeutic agents.

• Functional knockdown validation: Combine PAWR antibody-based detection with siRNA/shRNA knockdown or CRISPR-Cas9 knockout of PAWR to validate specificity and establish clear structure-function relationships in apoptotic cascades.

This multifaceted approach allows researchers to dissect PAWR's selective pro-apoptotic activity in cancer cells while sparing normal cells, potentially revealing novel therapeutic vulnerabilities .

What is the significance of PAWR-PITX2 interaction in ocular development and how can antibodies help investigate this?

The interaction between PAWR and PITX2 represents a significant molecular link between transcription factor regulation and apoptotic pathways in ocular development. Research using PAWR antibodies has revealed that:

• Interaction mechanisms: The homeodomain and adjacent inhibitory domain in PITX2 interact specifically with the C-terminal leucine zipper domain of PAWR. This interaction can be investigated using co-immunoprecipitation with anti-PAWR antibodies followed by immunoblotting for PITX2, or vice versa.

• Developmental co-localization: Both endogenous PAWR and PITX2 have been found to be located in the nucleus of ocular cells and co-localize in the mesenchyme of the iridocorneal angle during mouse eye development. This co-localization can be visualized through dual immunofluorescence staining using PAWR and PITX2 antibodies.

• Functional significance: PAWR has been found to inhibit PITX2 transcriptional activity in ocular cells, suggesting a regulatory role in eye development. This functional relationship can be investigated using:

  • Chromatin immunoprecipitation (ChIP) with PAWR antibodies to identify PAWR-PITX2 co-bound genomic regions

  • Reporter gene assays combined with immunoblotting to correlate PITX2 activity with PAWR expression levels

  • Immunohistochemistry in developmental time series to track the temporal relationship between these proteins

• Disease relevance: This interaction provides insight into the pathogenesis of Axenfeld-Rieger Syndrome (ARS) and associated glaucoma, where PITX2 mutations play a causal role. PAWR antibodies can be used to assess how disease-causing PITX2 mutations affect PAWR binding and localization.

This research direction connects developmental biology with apoptosis regulation and has implications for understanding congenital eye disorders .

How can advanced antibody engineering techniques improve PAWR antibody design for research applications?

Recent advances in antibody engineering can significantly enhance PAWR antibody design through computational and experimental approaches:

• Deep learning prediction models: Implementing neural network algorithms that analyze sequence-structure relationships can predict mutations that improve antibody specificity and affinity for PAWR epitopes. These models can be trained on existing antibody-antigen complex structures to generate optimized binding interfaces.

• Multi-objective linear programming approach: This computational method enables the design of antibody libraries with:

  • Optimized binding affinity to specific PAWR domains

  • Enhanced developability properties (solubility, stability)

  • Maximum diversity to ensure broad epitope coverage

• Structure-guided epitope selection: Using structural data of PAWR protein domains (particularly the C-terminal leucine zipper and N-terminal regions) to design antibodies targeting functionally significant epitopes that:

  • Distinguish between different PAWR conformational states

  • Specifically recognize post-translational modifications

  • Target protein-protein interaction surfaces

• Constraint parameters for optimization: When designing PAWR antibody libraries, constraints should be implemented including:

  • Minimum and maximum number of mutations from wild-type (n_min and n_max)

  • Position-specific diversity constraints

  • Mutation-specific representation limits

• Validation methods: Advanced antibody designs should undergo rigorous validation through:

  • Computational simulations of binding energetics

  • Surface plasmon resonance measurements of binding kinetics

  • Cell-based functional assays for epitope accessibility

This integrated approach combines deep learning with constrained optimization to create diverse, high-performing PAWR antibody libraries without requiring iterative experimental feedback, representing a "cold-start" design methodology that can accelerate research tool development .

What are common issues when using PAWR antibodies and how can they be resolved?

When working with PAWR antibodies, researchers may encounter several technical challenges that can be addressed through specific optimization strategies:

For optimal results with PAWR antibodies, validation using positive control tissues (such as human colon cancer tissue) and multiple detection methods is strongly recommended .

How should researchers interpret contradictory PAWR antibody results across different experimental systems?

When faced with contradictory results using PAWR antibodies across different experimental systems, researchers should conduct a systematic analysis following these methodological steps:

• Antibody validation assessment:

  • Confirm antibody specificity using knockout/knockdown controls

  • Test multiple PAWR antibodies targeting different epitopes

  • Assess batch-to-batch variation by requesting lot-specific validation data

• Context-dependent expression analysis:

  • PAWR expression and localization can vary significantly between tissue types and cellular conditions

  • Evaluate cell line differences systematically (cancer vs. normal, tissue origin)

  • Consider stress conditions that may affect PAWR expression (serum starvation, confluency)

• Technical parameter reconciliation:

  • Standardize protein extraction methods (RIPA vs. NP-40 buffers yield different results)

  • For fixation-sensitive epitopes, compare paraformaldehyde vs. methanol fixation

  • Adjust antigen retrieval methods for FFPE samples (citrate vs. EDTA buffers)

• Biological interpretation framework:

  • PAWR functions differently in cancer vs. normal cells

  • Nuclear vs. cytoplasmic localization correlates with different functions

  • Post-translational modifications may affect antibody recognition

• Integrated validation approach:

  • Complement antibody-based detection with mRNA analysis

  • Perform functional assays to correlate PAWR levels with known activities

  • Use genetic tools (CRISPR-Cas9) to validate antibody specificity

By systematically addressing these factors, researchers can reconcile seemingly contradictory results and develop a more nuanced understanding of PAWR biology across different experimental contexts .

What considerations are important when selecting a PAWR antibody for novel research applications?

When selecting a PAWR antibody for novel research applications, researchers should consider a comprehensive set of technical and experimental factors:

• Epitope mapping and domain recognition:

  • Determine which PAWR domain is most relevant to your research question

  • C-terminal antibodies (targeting the leucine zipper domain) are optimal for studying PITX2 interactions

  • N-terminal antibodies may better detect regulatory post-translational modifications

  • Middle region antibodies often provide robust detection across multiple applications

• Species cross-reactivity requirements:

  • Assess evolutionary conservation of target epitope if working across species

  • Confirm experimental validation in your species of interest

  • Consider developing custom antibodies for poorly conserved regions

• Application-specific performance metrics:

  • For protein-protein interaction studies: prioritize antibodies validated for immunoprecipitation

  • For localization studies: select antibodies with demonstrated performance in IF/ICC

  • For quantitative analysis: choose antibodies validated for linearity of signal

• Format and conjugation considerations:

  • Unconjugated antibodies offer maximum flexibility

  • Direct fluorophore conjugates reduce background in multi-color IF studies

  • HRP-conjugated versions may enhance sensitivity in certain applications

• Validation depth assessment:

  • Evaluate the breadth of validation data available (number of techniques and cell types)

  • Review independent literature citations using the antibody

  • Consider antibodies validated against recombinant PAWR protein standards

• Technical specifications for specialized applications:

  • For super-resolution microscopy: higher affinity antibodies may be required

  • For chromatin immunoprecipitation: confirms epitope accessibility in crosslinked samples

  • For multiplex assays: verify absence of cross-reactivity with other targets

By systematically evaluating these criteria, researchers can select the most appropriate PAWR antibody for their specific experimental needs and research questions .

How might PAWR antibodies contribute to understanding the role of PAWR in neurodegenerative diseases?

PAWR antibodies can be instrumental in elucidating PAWR's role in neurodegenerative pathologies through several methodological approaches:

• Comprehensive expression profiling: Using PAWR antibodies in immunohistochemistry and Western blotting to create an atlas of PAWR expression across neurodegenerative disease progression in both human post-mortem samples and animal models. This would include:

  • Quantitative analysis of PAWR levels in affected vs. unaffected brain regions

  • Correlation with markers of neuronal stress and apoptosis

  • Comparison between different neurodegenerative conditions (Alzheimer's, Parkinson's, ALS)

• Cell type-specific localization: Employing dual immunofluorescence with PAWR antibodies and cell type-specific markers to determine:

  • Differential expression between neurons, astrocytes, and microglia

  • Subcellular redistribution during disease progression

  • Co-localization with pathological protein aggregates (e.g., amyloid plaques, tau tangles)

• Mechanistic pathway analysis: Using PAWR antibodies in combination with other molecular tools to investigate:

  • Interaction with key neurodegeneration-associated proteins through co-immunoprecipitation

  • Post-translational modifications specific to diseased states using phospho-specific antibodies

  • Differential binding partners in healthy versus diseased tissue

• Therapeutic target validation: Utilizing PAWR antibodies to assess the efficacy of experimental therapeutics by:

  • Monitoring changes in PAWR expression and localization after treatment

  • Correlating PAWR levels with functional outcomes

  • Developing antibody-based imaging probes for in vivo assessment

• Biomarker development: Evaluating PAWR as a potential biomarker for neurodegenerative diseases through:

  • Quantitative immunoassays in CSF or plasma

  • Correlation with disease progression and severity

  • Assessment of prognostic value

This multifaceted approach would leverage the specificity of PAWR antibodies to connect PAWR's known pro-apoptotic functions with the pathophysiology of neurodegenerative disorders, potentially revealing novel therapeutic targets .

What emerging technologies might enhance the specificity and utility of PAWR antibodies for complex tissue analysis?

Several cutting-edge technologies are positioned to dramatically enhance PAWR antibody applications in complex tissue analysis:

• Spatial transcriptomics integration:

  • Combining PAWR antibody immunostaining with spatial transcriptomics to correlate protein expression with transcriptional profiles at single-cell resolution

  • Implementing computational frameworks to integrate protein and RNA data from the same tissue section

  • Developing multiplexed approaches to simultaneously visualize PAWR protein and mRNA

• Advanced multiplexing technologies:

  • Cyclic immunofluorescence (CycIF) or CO-Detection by indEXing (CODEX) to analyze PAWR alongside dozens of other proteins in the same tissue section

  • Metal-tagged antibodies for mass cytometry imaging (IMC) allowing simultaneous detection of >40 proteins with subcellular resolution

  • DNA-barcoded antibody technologies enabling ultra-high-plex protein mapping

• Nanobody and aptamer alternatives:

  • Development of PAWR-specific nanobodies (VHH fragments) for improved tissue penetration and reduced background

  • RNA or DNA aptamers targeting PAWR with high specificity and reduced immunogenicity

  • Bispecific binding molecules combining PAWR recognition with secondary target binding

• Artificial intelligence-enhanced analysis:

  • Deep learning algorithms for automated quantification of PAWR expression patterns across tissue sections

  • Convolutional neural networks to identify novel PAWR-associated tissue microenvironments

  • Predictive modeling to correlate PAWR spatial distribution with disease outcomes

• Live-cell and in vivo imaging adaptations:

  • Cell-permeable PAWR antibody derivatives for live-cell imaging

  • Near-infrared fluorophore-conjugated antibodies for in vivo PAWR tracking

  • Intrabodies derived from PAWR antibodies for real-time monitoring of PAWR dynamics

These technologies would transform PAWR antibody applications from traditional detection methods to dynamic, systems-level analysis of PAWR biology in intact tissues, providing unprecedented insights into its role in both physiological and pathological contexts .