PIK3R2 Antibody

What is PIK3R2 and why is it important in cell signaling research?

PIK3R2 encodes the p85β regulatory subunit of Class IA phosphoinositide 3-kinase (PI3K), a critical component in cell signaling pathways. This protein binds to activated (phosphorylated) protein-tyrosine kinases through its SH2 domain and functions as an adapter that mediates the association of the p110 catalytic unit to the plasma membrane . The PI3K signaling pathway coordinates fundamental cellular processes including cell growth, cell cycle entry, cell migration, and cell survival . Importantly, PIK3R2 has distinctive roles from other PI3K regulatory subunits, as evidenced by isoform-specific functions such as endocytosis being β isoform specific . Understanding PIK3R2 function is particularly significant because mutations in this gene have been linked to brain overgrowth syndromes, including megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome .

What applications are PIK3R2 antibodies validated for?

PIK3R2 antibodies have been validated for multiple experimental applications:

For optimal results, researchers should validate each antibody in their specific experimental system, as performance can vary based on tissue type, fixation method, and protein expression levels .

What are the recommended storage and handling protocols for PIK3R2 antibodies?

Proper storage and handling are critical for maintaining antibody activity and reproducibility:

• Long-term storage: Store at -20°C or -80°C in small aliquots (20 μl minimum) to prevent repeated freeze-thaw cycles

• Short-term use: For frequent use within 1-2 weeks, store at 4°C

• For concentrate or bioreactor products, consider adding an equal volume of glycerol as a cryoprotectant

• Avoid repeated freeze-thaw cycles as they can significantly reduce antibody activity

• When diluting, use fresh buffers prepared with high-quality reagents

• All commercial antibodies contain preservatives (commonly sodium azide), which should be considered when designing experiments, especially those involving enzymatic assays

How can researchers differentiate between PIK3R2 (p85β) and other PI3K regulatory subunits in experimental settings?

Differentiating between PI3K regulatory subunits requires careful experimental design:

• Antibody selection: Choose antibodies targeting non-conserved regions. For instance, antibodies targeting the N-terminal region of PIK3R2 show higher specificity than those targeting SH2 domains, which are more conserved across isoforms .

• Functional assays: The iSH2 domain from p85β (both human and mouse) induces endocytosis, but α or γ isoforms do not, providing a functional readout for isoform specificity . This can be validated using inducible co-recruitment assays with fluorescent markers.

• Mutation-specific approaches: For specific mutations like PIK3R2 p.G373R (p.G367R in mice), custom antibodies or genetic approaches may be necessary .

• Expression analysis: Quantitative comparisons of expression levels between different PI3K regulatory subunits can be achieved using standardized western blotting or qPCR approaches with appropriate normalization controls.

• Domain-specific interactions: The β isoform has distinct protein interaction partners. For example, PI3K p85β specifically interacts with AP2 complex through its iSH2 domain, which can be detected via pulldown assays .

What methodological approaches should be used when studying PIK3R2 mutations associated with neurological disorders?

Investigating PIK3R2 mutations in neurological disorders requires multiple complementary approaches:

• Genetic validation: Confirm mutations using genomic DNA extraction, PCR amplification, and sequencing. For the PIK3R2 c.265C > T (p.Arg89Cys) mutation associated with familial temporal lobe epilepsy, genomic DNA extraction using the Qiagen FlexiGene DNA kit followed by PCR (95°C for 10 min, 35 cycles at 95°C for 30s, 60°C for 30s, 72°C for 45s, and final extension at 72°C for 5 min) has been validated .

• Structural modeling: Use platforms like I-TASSER and PyMOL molecular graphics software to predict structural changes caused by mutations. Effects on protein function can be assessed with bioinformatics tools such as PolyPhen-2, SIFT, Mutation Taster, and CADD .

• Animal models: CRISPR/Cas9-generated knock-in mouse models (e.g., Pik3r2 p.G367R) provide valuable insights into in vivo pathogenesis. These models exhibit features similar to human patients, including brain overgrowth and seizure activity .

• Cellular reprogramming: Patient-derived induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells can be generated using non-integrating vector systems (e.g., CytoTune-iPS 2.0 Sendai Reprogramming Kit). After vector elimination (confirmed after passage 7), iPSCs can be characterized for pluripotency markers and karyotype stability .

• Functional assays: Assess PI3K pathway activation through phosphorylation of downstream targets like AKT and GSK-3β using phospho-specific antibodies in western blotting .

How can protein-protein interactions involving PIK3R2 be effectively studied?

Investigating PIK3R2 protein interactions requires multiple complementary approaches:

• Co-immunoprecipitation: Use PIK3R2-specific antibodies to pull down protein complexes, followed by western blotting or mass spectrometry to identify interaction partners. This approach has successfully identified interactions between PIK3R2 and proteins like IRS4 .

• Domain mapping: Create truncation constructs of PIK3R2 to identify minimal binding domains. For example, the C-terminal region (447-822aa, containing SH2 and kinase domains) of FER kinase showed robust interaction with IRS4, while the N-terminal region (1-446aa, F-BAR and FX domains) did not .

• Direct binding assays: Use purified proteins in pulldown assays to confirm direct interactions. GST-fused iSH2 domain pulldowns with AP2 core complex demonstrated direct interaction, which was disrupted by specific mutations .

• Inducible co-recruitment assays: Apply chemically inducible dimerization schemes to recruit PIK3R2 domains to specific cellular compartments and measure co-recruitment of suspected binding partners using fluorescence microscopy .

• Protein-protein interaction networks: Utilize databases like STRING and visualization tools like Cytoscape to map interaction networks. This approach has revealed connections between PIK3R2 and 32 temporal lobe epilepsy-related genes .

What are the critical controls and validation methods for PIK3R2 antibody experiments?

Rigorous validation is essential for reliable PIK3R2 antibody experiments:

• Antibody specificity controls:

  • Positive controls: Cell lines with known PIK3R2 expression (verified by western blot)

  • Negative controls: siRNA/shRNA knockdown or CRISPR knockout of PIK3R2

  • Pre-absorption controls: Pre-incubation with immunizing peptide should abolish specific signal

• Application-specific controls:

  • For Western blotting: Include molecular weight markers and loading controls

  • For IHC/ICC: Include isotype controls and secondary antibody-only controls

  • For IP experiments: Include IgG control pulldowns

• Cross-validation approaches:

  • Use multiple antibodies targeting different epitopes of PIK3R2

  • Compare results across different detection methods (e.g., WB, IHC, IF)

  • Correlate protein detection with mRNA expression data

• Documentation of validation data:

  • Commercial antibodies should provide validation data for stated applications

  • Antibody Registry IDs (e.g., AB_2617838 for AFFN-PIK3R2-2B2) aid in tracking validation history

• Functional validation:

  • Correlate antibody detection with functional readouts such as PI3K pathway activation

  • Use pathway inhibitors (e.g., LY294002) as control conditions

How can researchers optimize western blotting protocols for PIK3R2 detection?

Western blotting for PIK3R2 detection requires careful optimization:

• Sample preparation:

  • For cultured cells: Lysis in buffer containing protease and phosphatase inhibitors

  • For tissue samples: Homogenization in cold lysis buffer, followed by centrifugation to remove debris

  • Protein concentration determination by BCA or Bradford assay

• Gel electrophoresis considerations:

  • Use 8-10% SDS-PAGE gels for optimal resolution of PIK3R2 (predicted MW: 81.5 kDa)

  • Load adequate protein (typically 20-50 μg of total protein)

  • Include appropriate molecular weight markers

• Transfer optimization:

  • For PIK3R2, semi-dry or wet transfer protocols are suitable

  • Transfer time and voltage should be optimized for complete transfer of proteins in the 80-85 kDa range

• Antibody incubation:

  • Primary antibody dilution: 1:500-2000 depending on the specific antibody

  • Overnight incubation at 4°C often yields better results than shorter incubations

  • Thorough washing steps are critical for reducing background

• Signal detection:

  • Both chemiluminescence and fluorescence-based detection systems are compatible

  • For weak signals, consider signal enhancement systems or longer exposure times

• Troubleshooting common issues:

  • High background: Increase blocking time/concentration or add 0.05% Tween-20 to washing buffer

  • No signal: Check protein transfer efficiency with reversible staining

  • Multiple bands: Validate with alternative antibodies or knockdown controls

What considerations are important when designing experiments to study PIK3R2 in disease models?

When investigating PIK3R2 in disease models, several critical considerations should be addressed:

• Model selection:

  • Cell lines: Choose models with appropriate PIK3R2 expression levels

  • Animal models: Consider knock-in models carrying specific mutations (e.g., Pik3r2 p.G367R for MPPH syndrome)

  • Patient-derived samples: iPSCs can be generated from patient PBMCs to study disease-relevant phenotypes

• Experimental readouts:

  • Morphological analysis: Brain size measurements in animal models

  • Cellular phenotypes: Cell size, proliferation, and migration assays

  • Molecular markers: PI3K pathway activation (phospho-AKT levels)

  • Functional assessments: EEG recordings for seizure activity in epilepsy models

• Developmental timing:

  • For neurodevelopmental disorders, analyze multiple developmental stages

  • In the Pik3r2 p.G367R mouse model, embryonic brain showed mild defects in cortical lamination not observed in mature brain

• Data analysis frameworks:

  • Compare genotype-phenotype correlations across different models

  • Integrate multiple readouts (cellular, molecular, functional)

  • Consider incomplete penetrance in hereditary conditions

• Translational relevance:

  • Correlate findings in model systems with patient data

  • Consider therapeutic implications (e.g., PI3K pathway inhibitors)

  • Acknowledge limitations of models in recapitulating human disease complexity

How can researchers effectively use PIK3R2 antibodies across different species?

Cross-species applications of PIK3R2 antibodies require careful consideration:

• Sequence homology assessment:

  • Compare PIK3R2 sequences across target species

  • Focus on antibodies targeting highly conserved epitopes for cross-species applications

• Validated species reactivity:

  • Commercial antibodies report species reactivity based on validation data

  • For example, AFFN-PIK3R2-2B2 is validated for human samples

  • Anti-PI3 Kinase p85 beta PIK3R2 Monoclonal Antibody (M03363-1) is validated for human, mouse, and rat

  • PIK3R2 Antibody - N-terminal region (OAAB17389) reports reactivity with bovine, human, mouse, and rat

• Application-specific adjustments:

  • Antibody concentration may need adjustment for different species

  • Incubation times and temperatures may require optimization

  • For IHC, tissue fixation protocols may need species-specific modifications

• Validation strategies:

  • Positive controls from each species should confirm specificity

  • Western blot should show bands of appropriate molecular weight

  • For novel applications, preliminary validation is essential

• Isoform considerations:

  • Species may express different PIK3R2 isoforms or splice variants

  • Antibodies targeting specific isoforms may not work across species

  • Document exact protein isoform detected in each species

How can PIK3R2 antibodies be applied in studying the PI3K-AKT-mTOR pathway activation?

PIK3R2 antibodies serve as valuable tools for investigating the PI3K-AKT-mTOR pathway:

• Pathway activation analysis:

  • Combined detection of PIK3R2 and phosphorylated downstream effectors (p-AKT, p-GSK-3β, p-mTOR)

  • Quantitative western blotting with phospho-specific antibodies can measure relative pathway activation

  • In the Pik3r2 p.G367R mouse model, hyperactivation of the PI3K-AKT pathway confirmed the mutation's activating nature in vivo

• Inhibitor studies:

  • PIK3R2 antibodies can monitor protein levels during treatment with pathway inhibitors

  • LY294002 binds to the ATP binding pocket of p110 and inhibits catalytic function, providing a tool to dissect PI3K-dependent and independent functions

  • Combined detection of PIK3R2 and pathway components during inhibitor treatment reveals regulatory relationships

• Subcellular localization:

  • Immunofluorescence with PIK3R2 antibodies reveals localization patterns

  • Co-localization with phosphorylated effectors indicates sites of active signaling

  • Changes in localization during stimulation provide insights into activation mechanisms

• Disease model applications:

  • In MPPH syndrome, PIK3R2 mutations cause excessive activation of PI3K/AKT/mTOR pathway

  • Antibodies help quantify this hyperactivation and evaluate potential therapeutic interventions

  • Comparative analysis between normal and disease states identifies critical regulatory nodes

• Temporal dynamics:

  • Time-course experiments with PIK3R2 antibodies reveal dynamics of pathway activation

  • Sequential phosphorylation events can be mapped to understand signaling cascades

  • Correlation with functional outcomes provides physiologically relevant insights

What methodological approaches are recommended for studying PIK3R2 mutations in brain development disorders?

Studying PIK3R2 mutations in brain development disorders requires integrated approaches:

• Genetic and molecular characterization:

  • Genomic DNA extraction and targeted sequencing of PIK3R2 and related genes

  • Bioinformatic analysis using tools like PolyPhen-2, SIFT, and Mutation Taster to predict pathogenicity

  • Structure modeling using I-TASSER and PyMOL to visualize mutation effects on protein structure

• Cellular phenotyping:

  • Analysis of cell size, number, and density in animal models

  • In Pik3r2 p.G367R mice, cell size was increased without changes in cell numbers

  • Evaluation of cortical lamination and interneuron migration in developmental studies

• Functional assessment:

  • EEG recordings to detect seizure activity and background slowing

  • Behavioral testing for cognitive function

  • Correlation of phenotypes with human patient data

• Pathway analysis:

  • Western blotting with phospho-specific antibodies to measure PI3K-AKT pathway activation

  • Protein-protein interaction network construction using databases like STRING and visualization with Cytoscape

  • Integration of PIK3R2 interactions with disease-specific gene networks

• Therapeutic exploration:

  • Testing pathway inhibitors in cellular and animal models

  • Monitoring treatment effects on both molecular markers and functional outcomes

  • Using PIK3R2 antibodies to track target engagement and pathway modulation

This integrated approach has successfully characterized PIK3R2 mutations in conditions like MPPH syndrome and familial temporal lobe epilepsy, providing insights into pathogenic mechanisms and potential therapeutic targets .

How are PIK3R2 antibodies used in epilepsy research?

PIK3R2 antibodies have become valuable tools in epilepsy research following the discovery of PIK3R2 mutations in epilepsy patients:

• Expression analysis in epileptic tissue:

  • Immunohistochemistry using PIK3R2 antibodies has revealed significantly higher expression of PIK3R2 in resected temporal lobe cortex from patients with refractory temporal lobe epilepsy compared to non-epileptic controls

  • Western blotting quantification provides numerical data on expression differences

• Animal model validation:

  • In Pik3r2 p.G367R mouse models, EEG recordings showed background slowing and rare seizures, similar to observations in human patients

  • PIK3R2 antibodies help confirm the molecular phenotype underlying these electrophysiological changes

• Mechanistic studies:

  • Protein-protein interaction networks constructed using co-immunoprecipitation with PIK3R2 antibodies have identified connections between PIK3R2 and 32 temporal lobe epilepsy-related genes

  • Pathway analysis reveals how PIK3R2 mutations affect downstream signaling in epilepsy

• Biomarker development:

  • Expression patterns detected by PIK3R2 antibodies may serve as diagnostic or prognostic biomarkers

  • Correlation of PIK3R2 levels with seizure frequency or treatment response provides clinical insights

• Therapeutic target validation:

  • PIK3R2 antibodies help monitor the effects of PI3K pathway inhibitors in pre-clinical epilepsy models

  • Changes in PIK3R2 expression or interaction patterns may predict treatment efficacy

What are the key experimental considerations when using PIK3R2 antibodies in cancer research?

PIK3R2 antibodies are important tools in cancer research due to the role of the PI3K pathway in oncogenesis:

• Expression analysis across cancer types:

  • Western blotting and IHC with PIK3R2 antibodies can map expression patterns across tumor types

  • Correlation with clinical outcomes provides prognostic insights

  • Comparison with normal tissue identifies cancer-specific alterations

• Pathway activation assessment:

  • Combined detection of PIK3R2 and phosphorylated downstream components (p-AKT, p-mTOR)

  • The PI3K signaling pathway is constitutively activated in human cancers with loss of function of PTEN

  • PIK3R2 antibodies help differentiate between different mechanisms of pathway activation

• Interaction studies in cancer contexts:

  • Co-immunoprecipitation with PIK3R2 antibodies can identify cancer-specific interaction partners

  • Changes in regulatory interactions may explain altered signaling in cancer cells

  • The interaction between p85β and catalytic p110 subunits can be monitored using appropriate antibodies

• Response to targeted therapies:

  • PIK3R2 antibodies track molecular changes during treatment with PI3K pathway inhibitors

  • Expression and phosphorylation patterns may predict response to targeted therapies

  • Sequential biopsies during treatment provide insights into resistance mechanisms

• Technical considerations in cancer samples:

  • Tissue heterogeneity requires careful analysis of PIK3R2 expression patterns

  • Patient-derived samples may have variable preservation quality affecting antibody performance

  • Correlation with genetic data (mutations, copy number variations) provides comprehensive characterization