PIK3CB Antibody

What is PIK3CB and why is it significant in cellular research?

PIK3CB (phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform) is a 1070 amino acid protein belonging to the PI3/PI4-kinase family. It functions as a catalytic subunit that phosphorylates PtdIns (phosphatidylinositol), PtdIns4P, and PtdIns(4,5)P2 to generate phosphatidylinositol 3,4,5-triphosphate (PIP3) . This enzyme plays crucial roles in signaling pathways regulating cell growth, survival, proliferation, motility, and morphology by activating downstream proteins containing PH domains . PIK3CB is one of four class I catalytic subunits (p110α, p110β, p110δ, and p110γ) that associate with regulatory adaptor proteins to form heterodimeric PI3 kinases . Its significance lies in its central role in PI3K signaling pathways implicated in various physiological and pathological processes, including cancer development.

What types of PIK3CB antibodies are available for research applications?

Research-grade PIK3CB antibodies are available in several formats:

Recombinant rabbit monoclonal antibodies offer particular advantages for research applications as they are produced using in vitro expression systems after cloning antibody DNA sequences from immunoreactive rabbits . This production method ensures greater consistency between batches and broader immunoreactivity due to the larger immune repertoire of rabbits .

What are the validated applications for PIK3CB antibodies?

PIK3CB antibodies have been validated for multiple applications across different experimental systems:

For optimal results, it is recommended that each antibody be titrated in the specific testing system being used, as performance can be sample-dependent .

How should I optimize Western blot protocols for PIK3CB detection?

When optimizing Western blot protocols for PIK3CB detection, consider these methodological steps:

• Sample preparation: Use RIPA buffer with protease and phosphatase inhibitors for effective protein extraction

• Loading amount: Load 20-50 μg of total protein per lane for cell lysates

• Separation: Use 8-10% SDS-PAGE gels for optimal resolution of the 110-130 kDa PIK3CB protein

• Transfer: Employ wet transfer at 100V for 60-90 minutes with methanol-containing buffer

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

• Primary antibody: Dilute PIK3CB antibody according to manufacturer's recommendation (typically 1:200-1:1000) and incubate overnight at 4°C

• Detection: Use appropriate HRP-conjugated secondary antibody and chemiluminescence detection

For specific antibodies like PAB3213, a dilution of 1:1000 has been validated for detecting PIK3CB in Jurkat cell lysates . The expected molecular weight observed on Western blots is typically between 110-130 kDa .

What are the critical factors for successful immunohistochemistry with PIK3CB antibodies?

Successful immunohistochemistry (IHC) with PIK3CB antibodies requires attention to several critical factors:

• Tissue fixation and processing: Use 10% neutral buffered formalin for fixation and standard paraffin embedding

• Antigen retrieval: For optimal results, perform heat-induced epitope retrieval with TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 can be used

• Antibody dilution: For IHC applications, dilute primary antibody within the recommended range (1:50-1:500)

• Incubation conditions: Incubate sections with primary antibody overnight at 4°C in a humidified chamber

• Detection system: Use polymer-based or avidin-biotin detection systems with appropriate chromogenic substrates (DAB is commonly used)

• Counterstaining: Lightly counterstain with hematoxylin to visualize tissue architecture

• Controls: Always include positive control tissues (human lung cancer, mouse brain, or human prostate carcinoma have been validated)

PIK3CB antibodies have been successfully applied to formalin-fixed, paraffin-embedded human prostate carcinoma tissues using peroxidase-conjugated secondary antibodies followed by DAB staining .

How can I use PIK3CB antibodies to investigate PI3K signaling in cancer models?

Investigating PI3K signaling in cancer models with PIK3CB antibodies involves several sophisticated approaches:

• Isoform-specific signaling analysis:

  • Use PIK3CB-specific antibodies alongside other PI3K isoform antibodies (p110α, p110δ, p110γ) to determine the predominant isoforms in different cancer types

  • Compare expression patterns across cancer cell lines and patient-derived xenografts using Western blot and IHC

• Subcellular localization studies:

  • Employ confocal microscopy with PIK3CB antibodies (dilution 1:200-1:800) for immunofluorescence

  • Analyze co-localization with membrane markers, endosomal markers, or nuclear proteins to determine compartment-specific functions

• Protein-protein interaction networks:

  • Utilize co-immunoprecipitation with PIK3CB antibodies (0.5-4.0 μg for 1.0-3.0 mg protein)

  • Combine with proximity ligation assays to visualize and quantify protein interactions in situ

  • Map the interactome of PIK3CB in different cancer contexts

• Functional studies with genetic manipulation:

  • Validate antibody specificity using PIK3CB knockdown/knockout models

  • Monitor changes in PIK3CB expression/localization following treatment with PI3K inhibitors

This multi-faceted approach allows researchers to comprehensively characterize the role of PIK3CB in cancer biology and potentially identify new therapeutic targets or biomarkers.

What approaches can resolve discrepancies between PIK3CB protein detection and functional activity?

Resolving discrepancies between PIK3CB protein detection and functional activity requires sophisticated experimental design:

• Antibody epitope considerations:

  • Different antibodies recognize distinct epitopes that may be masked by protein interactions or post-translational modifications

  • Use multiple antibodies targeting different regions of PIK3CB (N-terminal, catalytic domain, C-terminal) to obtain a complete picture

  • For instance, antibody 1H9L37 recognizes a different epitope than 20584-1-AP

• Assessment of post-translational modifications:

  • Complement standard Western blots with phospho-specific antibodies

  • Perform immunoprecipitation with PIK3CB antibodies followed by mass spectrometry to identify modifications

• Kinase activity assays:

  • Correlate protein detection with functional PIP3 production using specialized assays

  • Consider competitive vs. non-competitive inhibition mechanisms when evaluating inhibitor studies

• Protein complex integrity analysis:

  • Assess regulatory subunit (p85) association using co-immunoprecipitation

  • Investigate the integrity of higher-order signaling complexes that might affect kinase activity

• Feedback regulation mechanisms:

  • Examine temporal dynamics of PIK3CB expression and activity

  • Investigate compensatory upregulation of other PI3K isoforms

This comprehensive approach helps reconcile apparent contradictions between protein levels and functional outcomes, providing deeper insights into context-dependent PIK3CB regulation.

How can I address non-specific binding when using PIK3CB antibodies?

Non-specific binding is a common challenge when working with PIK3CB antibodies. Here's a methodological approach to address this issue:

• Antibody selection and validation:

  • Choose antibodies with validated specificity for PIK3CB, such as recombinant monoclonal antibodies that offer better specificity

  • Verify antibody specificity using positive control samples (rat liver tissue, HepG2 cells)

  • Include negative controls such as isotype controls and PIK3CB-knockdown samples

• Blocking optimization:

  • Test different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Perform titration experiments to determine optimal antibody concentration

  • For Western blot, test dilutions from 1:200 to 1:1000

  • For IHC, begin with 1:50 and adjust as needed up to 1:500

  • Extend primary antibody incubation to overnight at 4°C with gentle agitation

• Washing stringency:

  • Increase number of wash steps (minimum 3-5 washes)

  • Extend washing time to 10 minutes per wash

  • Add 0.05-0.1% Tween-20 to wash buffers

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Dilute secondary antibodies appropriately (typically 1:2000-1:10000)

  • Consider using fluorescent secondary antibodies for better signal-to-noise ratio

By systematically optimizing these parameters, researchers can significantly reduce non-specific binding and improve the quality of their PIK3CB detection assays.

What are the best practices for storing and handling PIK3CB antibodies to maintain optimal activity?

Proper storage and handling of PIK3CB antibodies is crucial for maintaining their activity and specificity over time:

Best practices for handling:

• Always wear gloves to prevent contamination

• Avoid repeated freeze-thaw cycles by preparing appropriate aliquots

• Bring antibodies to room temperature before opening to prevent condensation

• Centrifuge vials briefly before opening to collect liquid at the bottom

• Return antibodies to appropriate storage immediately after use

• Note that some PIK3CB antibody preparations contain sodium azide, which is toxic and should be handled with appropriate precautions

For recombinant antibodies like 1H9L37, follow manufacturer's specific storage recommendations to maintain the advantages of lot-to-lot consistency . For antibodies in glycerol solutions like 20584-1-AP, aliquoting is unnecessary for -20°C storage in the 20μL size containing 0.1% BSA .

How can PIK3CB antibodies be utilized in multiplexed imaging systems for tumor microenvironment analysis?

PIK3CB antibodies can be leveraged in cutting-edge multiplexed imaging approaches for comprehensive tumor microenvironment analysis:

• Multiplex immunofluorescence (mIF):

  • Combine PIK3CB antibodies with markers for immune cells, stromal components, and other signaling proteins

  • Implement sequential staining protocols using tyramide signal amplification

  • Develop antibody panels that include PIK3CB (1:200-1:800 dilution) alongside markers like CD45, CD8, PDL1, and phospho-AKT

• Imaging mass cytometry (IMC):

  • Conjugate PIK3CB antibodies with rare earth metals

  • Analyze spatial distribution of up to 40 markers simultaneously on the same tissue section

  • Create detailed maps of PIK3CB expression in relation to the cellular landscape

• Cyclic immunofluorescence (CycIF):

  • Incorporate PIK3CB antibodies into iterative staining/imaging/quenching workflows

  • Build high-dimensional datasets revealing PIK3CB distribution in the spatial context of numerous cell types

• Digital spatial profiling:

  • Apply PIK3CB antibodies in conjunction with region-specific oligonucleotide barcodes

  • Correlate protein expression with transcriptomic data in defined tissue regions

These advanced imaging approaches allow researchers to examine the heterogeneity of PIK3CB expression across different cell populations within the tumor microenvironment, providing insights into its role in tumor-stroma interactions and immune modulation.

What considerations are important when developing phospho-specific antibodies for PIK3CB?

Developing phospho-specific antibodies for PIK3CB requires attention to several critical factors:

• Phosphorylation site selection:

  • Identify functionally relevant phosphorylation sites through kinase prediction algorithms and mass spectrometry

  • Focus on sites with known regulatory functions or those conserved across species

  • Consider sites that change dynamically with cellular stimulation or inhibition

• Peptide design strategy:

  • Design immunogenic phosphopeptides that include 8-20 amino acids surrounding the phosphorylation site

  • Ensure the phosphopeptide is unique to PIK3CB to avoid cross-reactivity with other PI3K family members

  • Include a carrier protein (KLH is commonly used) for peptide conjugation, similar to the approach used for existing PIK3CB antibodies

• Antibody screening and validation:

  • Implement rigorous validation protocols using phosphatase-treated samples as negative controls

  • Verify phospho-specificity using site-directed mutagenesis (Ser/Thr to Ala)

  • Test antibody performance across multiple applications (Western blot, IHC, IF)

  • Validate reactivity across species if cross-species reactivity is desired

• Technical challenges to address:

  • Low abundance of phosphorylated forms may require signal amplification methods

  • Transient nature of phosphorylation requires careful timing of sample collection

  • Preservation of phosphorylation status during sample preparation is critical

• Application-specific considerations:

  • For Western blot applications, include phosphatase inhibitors in lysis buffers

  • For IHC applications, develop specialized fixation protocols that preserve phospho-epitopes

  • For IF applications, optimize permeabilization conditions to maintain phospho-epitope integrity

Developing well-characterized phospho-specific PIK3CB antibodies would significantly advance research into the regulatory mechanisms controlling this important signaling protein.

How can PIK3CB antibodies be used to evaluate the specificity of p110β inhibitors in preclinical models?

PIK3CB antibodies serve as essential tools for evaluating p110β inhibitor specificity in preclinical models through several methodological approaches:

• Target engagement assessment:

  • Use PIK3CB antibodies in cellular thermal shift assays (CETSA) to measure direct binding of inhibitors to p110β

  • Perform drug affinity responsive target stability (DARTS) assays with subsequent Western blot using PIK3CB antibodies (1:200-1:1000)

  • Implement biolayer interferometry or surface plasmon resonance with purified protein and validate results with PIK3CB antibodies

• Pathway selectivity analysis:

  • Monitor phosphorylation of downstream targets after inhibitor treatment

  • Compare effects on p110β-dependent vs. other PI3K isoform-dependent signaling

  • Use PIK3CB antibodies in co-immunoprecipitation studies to examine effects on regulatory interactions

• Combination with genetic approaches:

  • Validate inhibitor specificity using PIK3CB knockout/knockdown models

  • Compare phenotypic effects of genetic vs. pharmacological inhibition

  • Screen for potential off-target effects using proteomic approaches followed by validation with PIK3CB antibodies

• Cancer model evaluation:

  • Use IHC with PIK3CB antibodies (1:50-1:500) to analyze expression in patient-derived xenografts before and after inhibitor treatment

  • Correlate PIK3CB expression levels with sensitivity to p110β inhibitors

  • Investigate resistance mechanisms by examining PIK3CB localization and interaction changes

These approaches provide comprehensive assessment of inhibitor specificity and mechanism of action, crucial for advancing p110β-targeted therapeutics from preclinical to clinical development.

What methodological approaches can distinguish between PIK3CB isoform-specific functions in different tissue contexts?

Distinguishing PIK3CB isoform-specific functions across different tissue contexts requires sophisticated methodological approaches:

• Tissue-specific expression profiling:

  • Implement systematic IHC screening with PIK3CB antibodies across tissue microarrays

  • Quantify relative expression levels of p110β compared to other PI3K isoforms

  • For tissues like human lung cancer, mouse brain, and prostate carcinoma, use validated PIK3CB antibodies at 1:50-1:500 dilution

• Isoform-selective genetic models:

  • Develop tissue-specific PIK3CB conditional knockout models

  • Create knock-in models with catalytically inactive PIK3CB to distinguish scaffolding from enzymatic functions

  • Validate models using PIK3CB antibodies in Western blot (1:200-1:1000) and IHC applications

• Substrate specificity analysis:

  • Perform immunoprecipitation with PIK3CB antibodies (0.5-4.0 μg for 1.0-3.0 mg protein)

  • Couple with mass spectrometry to identify tissue-specific interaction partners

  • Use proximity ligation assays to visualize and quantify protein interactions in intact tissues

• Context-dependent signaling analysis:

  • Apply PIK3CB antibodies in multiplex immunofluorescence to simultaneously detect multiple signaling components

  • Compare signaling dynamics across multiple tissue types and disease states

  • Implement single-cell analysis techniques to address cellular heterogeneity within tissues

• Functional genomics approaches:

  • Conduct CRISPR screens with PIK3CB-related genes in tissue-specific cell models

  • Validate hits using PIK3CB antibodies to assess effects on protein expression and localization

  • Integrate findings with patient data to establish clinical relevance

These methodological approaches allow researchers to delineate the distinct roles of PIK3CB across different physiological and pathological contexts, providing insights into tissue-specific therapeutic targeting strategies.