PIK3R5 Antibody

What is PIK3R5 and why is it important in cell signaling research?

PIK3R5, also known as p101 or FOAP-2, is the regulatory subunit 5 of phosphoinositide 3-kinase (PI3K). It plays a crucial role in the PI3K signaling pathway, which is involved in cellular functions including growth, proliferation, differentiation, motility, survival, and intracellular trafficking. This pathway is implicated in cancer and other diseases, making PIK3R5 an important research target. The regulatory function of PIK3R5 comes from its interaction with PIK3CG, making it essential for proper PI3K function and downstream signaling effects . Understanding PIK3R5 is vital for researchers investigating cellular signaling mechanisms and potential therapeutic targets in disease states.

How do I select the appropriate PIK3R5 antibody clone for my specific research application?

When selecting a PIK3R5 antibody, consider several factors based on your experimental needs:

• Species reactivity: Determine which species your samples are from. Available antibodies show reactivity across human, mouse, rat, dog, and monkey species .

• Application compatibility: Different clones perform optimally in specific applications:

  • For Western blotting: Monoclonal antibodies like OTI4G9 (1:500-2000 dilution) or 2A6 offer good specificity

  • For immunohistochemistry: Select antibodies validated for IHC, typically used at 1:150 dilution

  • For immunofluorescence: Consider clones validated for IF applications (1:100 dilution)

• Epitope recognition: Depending on your research question, select antibodies targeting specific domains:

  • N-terminal targeting antibodies for structural studies

  • C-terminal targeting antibodies (such as those recognizing AA 763-792) for functional studies

  • Internal epitope antibodies for general detection

• Clonality preference: Monoclonal antibodies (like OTI4G9, 2A6, 5B1) provide consistent lot-to-lot reproducibility, while polyclonal antibodies may offer broader epitope recognition but with potential batch variation .

The optimal choice depends on your specific experimental goals, tissue types, and detection methods.

What are the primary applications for PIK3R5 antibodies in cell biology research?

PIK3R5 antibodies serve multiple critical applications in cell biology research:

• Western Blotting (WB): Enables quantification and molecular weight verification of PIK3R5 (theoretical MW 97.2 kDa). Useful for detecting expression levels across different cell lines and experimental conditions .

• Immunohistochemistry (IHC): Allows visualization of PIK3R5 expression patterns within tissue contexts, particularly important for in situ analysis of signaling pathway components in normal versus diseased states .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Enables subcellular localization studies to determine where PIK3R5 functions within cells and how its distribution changes under various stimuli or conditions .

• Protein-Protein Interaction Studies: Some antibodies are suitable for co-immunoprecipitation or proximity ligation assays (PLA) to investigate PIK3R5's interaction with binding partners like PIK3CG .

• Flow Cytometry (FACS): Select antibodies can be used to quantify PIK3R5 expression at the single-cell level, particularly useful for heterogeneous cell populations .

These applications collectively enable researchers to investigate PIK3R5's expression, localization, interactions, and functional significance in cellular signaling pathways.

What controls should I include when using PIK3R5 antibodies for immunocytochemistry experiments?

For rigorous immunocytochemistry experiments with PIK3R5 antibodies, incorporate these essential controls:

• Primary antibody specificity controls:

  • Positive control: Use cells with known PIK3R5 expression (e.g., HEK293T cells transfected with PIK3R5 expression vector as shown in validation studies)

  • Negative control: Use cells without primary antibody treatment but with secondary antibody to assess non-specific binding

  • Isotype control: Include matched isotype control antibody (e.g., Mouse IgG2b for clone 2A6) to identify any non-specific binding related to the antibody class

• Cross-reactivity considerations:

  • When using mouse-derived antibodies (like OTI4G9) on mouse tissue, implement Mouse-On-Mouse blocking reagents to reduce background signal

  • Consider testing on PIK3R5 knockout/knockdown cells to confirm specificity

• Technical controls:

  • Concentration gradient: Test antibody at multiple dilutions (starting with 1:100 as recommended)

  • Subcellular marker co-staining: Use established subcellular markers to confirm expected localization pattern

  • Fluorophore controls: Include single-color controls if performing multi-color immunofluorescence

• Signal validation:

  • Verify results with a second PIK3R5 antibody targeting a different epitope

  • Compare staining patterns with published literature on PIK3R5 localization

Careful implementation of these controls ensures reliable and interpretable immunocytochemistry results with minimal artifacts or false positives.

How should I optimize Western blot protocols specifically for PIK3R5 detection?

Optimizing Western blot protocols for PIK3R5 detection requires attention to several critical parameters:

• Sample preparation:

  • Use fresh lysates when possible and include protease inhibitors to prevent degradation

  • For cell lines with variable PIK3R5 expression, refer to validated positive controls such as HEK293T, HeLa, Jurkat, or MCF7 cells

  • Load sufficient protein (35μg has been validated across multiple cell lines)

• Gel selection and transfer:

  • Use 8-10% SDS-PAGE gels to properly resolve PIK3R5's 97.2 kDa molecular weight

  • Consider gradient gels if analyzing both PIK3R5 and binding partners of different sizes

  • Optimize transfer conditions for high molecular weight proteins (longer transfer time or lower current)

• Antibody incubation:

  • Initial dilution range: 1:500-2000 for PIK3R5 antibodies like OTI4G9

  • Incubate primary antibody overnight at 4°C to improve sensitivity

  • Consider 5% BSA rather than milk for blocking and antibody dilution if phosphorylated forms are being detected

• Detection and troubleshooting:

  • Be aware that observed molecular weight may vary from the predicted 97.2 kDa due to post-translational modifications

  • If detecting transfected tagged PIK3R5, account for the additional weight of the tag

  • For weak signals, increase protein load or reduce antibody dilution before extending exposure times

• Normalization and quantification:

  • Strip and reprobe for loading controls appropriate for your experimental context

  • Use housekeeping proteins expressed at comparable levels to PIK3R5 for accurate quantification

Following these optimization strategies will yield cleaner, more reproducible Western blot results for PIK3R5 detection across experimental conditions.

What reconstitution and storage protocols maximize PIK3R5 antibody stability and performance?

To maximize PIK3R5 antibody stability and performance, follow these evidence-based reconstitution and storage protocols:

• Initial reconstitution:

  • For lyophilized antibodies (like NBP2-73408), add 100μL distilled water to achieve approximately 1 mg/mL concentration

  • Allow complete dissolution at room temperature, with gentle rotation rather than vortexing

  • For conjugation experiments, consider an additional desalting step after reconstitution

• Storage conditions:

  • Store reconstituted antibodies at -20°C and avoid repeated freeze-thaw cycles

  • Aliquot antibodies into single-use volumes based on your typical experiment needs

  • For short-term use (within 1-2 weeks), antibodies can be stored at 4°C with appropriate preservatives

• Buffer considerations:

  • PIK3R5 antibodies are typically formulated in PBS (pH 7.3) with 8% trehalose

  • Note whether your antibody is azide-free, particularly important for certain applications like functional assays

  • For long-term storage, consider adding glycerol (final concentration 30-50%) to prevent freeze-damage

• Working solution preparation:

  • Prepare fresh working dilutions on the day of experimentation

  • Use buffers appropriate for your application (PBS with 0.1-0.5% BSA for most applications)

  • Document lot numbers, reconstitution dates, and dilution factors for reproducibility

• Performance monitoring:

  • Periodically test antibody performance against a reference sample

  • Consider preparing a standard positive control lysate in bulk and storing as single-use aliquots

  • Monitor for signs of degradation: reduced signal intensity, increased background, or appearance of non-specific bands

Adhering to these protocols will ensure optimal antibody performance and extend the useful life of your PIK3R5 antibodies across multiple experiments.

How can I utilize PIK3R5 antibodies to investigate protein-protein interactions in the PI3K signaling pathway?

Investigating PIK3R5 protein-protein interactions in the PI3K signaling pathway requires sophisticated approaches:

• Co-immunoprecipitation (Co-IP):

  • Select PIK3R5 antibodies that don't interfere with known binding regions, particularly the PIK3CG interaction interface

  • Use gentle lysis conditions (non-ionic detergents like NP-40 or Triton X-100 at 0.5-1%)

  • Optimize antibody:protein ratios to maximize capture without disrupting complexes

  • Validate results bidirectionally by performing reverse Co-IPs (e.g., pull down with PIK3CG antibody, probe for PIK3R5)

• Proximity Ligation Assay (PLA):

  • Several PIK3R5 antibodies are validated for PLA applications

  • Use antibody pairs from different host species (e.g., mouse anti-PIK3R5 and rabbit anti-PIK3CG)

  • Include distance controls (proteins known to be more distant than the PLA detection limit of ~40nm)

  • Quantify interaction signals using appropriate image analysis software

• Bimolecular Fluorescence Complementation (BiFC):

  • Use PIK3R5 antibodies to validate expression and localization of fusion constructs

  • Compare BiFC results with antibody-based detection of endogenous interactions

  • Perform competition assays with untagged PIK3R5 to confirm specificity

• Cross-linking Mass Spectrometry:

  • Use PIK3R5 antibodies for enrichment of cross-linked complexes prior to MS analysis

  • Validate MS-identified interactions using traditional antibody-based methods

  • Design experiments to capture both stable and transient interactions

• Experimental verification:

  • Employ domain-specific antibodies to map binding interfaces

  • Use phospho-specific antibodies to correlate phosphorylation status with interaction dynamics

  • Include physiologically relevant activators or inhibitors of the PI3K pathway to assess context-dependent interactions

These approaches, used complementarily, provide robust evidence for PIK3R5's interaction partners and their functional significance in PI3K signaling.

What are the common pitfalls when using PIK3R5 antibodies for immunohistochemistry on paraffin-embedded tissues?

Researchers frequently encounter several challenges when using PIK3R5 antibodies for immunohistochemistry on paraffin-embedded tissues:

• Epitope masking issues:

  • Formalin fixation can cross-link proteins and mask PIK3R5 epitopes

  • Solution: Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Test multiple retrieval conditions with positive control tissues

• Species cross-reactivity concerns:

  • When using mouse-derived PIK3R5 antibodies (like OTI4G9) on mouse tissues, high background is common

  • Solution: Implement Mouse-On-Mouse blocking reagents (e.g., those referenced as PK-2200-NB and MP-2400-NB)

  • Use antibodies raised in species different from your tissue source when possible

• Signal specificity verification:

  • PIK3R5 may show variable expression across different cell types within the same tissue

  • Solution: Include positive and negative control tissues with known PIK3R5 expression profiles

  • Validate staining patterns with antibodies targeting different PIK3R5 epitopes

• Optimal antibody dilution determination:

  • Starting dilution of 1:150 for PIK3R5 antibodies like OTI4G9 is recommended , but optimization is essential

  • Solution: Perform antibody titration experiments (1:50 to 1:500) on control tissues

  • Balance specific signal versus background for each tissue type

• Detection system selection:

  • PIK3R5's expression level may require signal amplification in some tissues

  • Solution: Compare standard two-step detection versus amplification systems (e.g., polymer-based or tyramide signal amplification)

  • Select detection systems compatible with multiplex staining if co-localization studies are planned

• Interpretation challenges:

  • Distinguishing specific from non-specific staining requires experience

  • Solution: Include isotype controls and secondary-only controls

  • Compare subcellular localization patterns with published data on PIK3R5

Addressing these pitfalls systematically will significantly improve the reliability and interpretability of PIK3R5 immunohistochemistry results in paraffin-embedded tissues.

How do I interpret conflicting results between different detection methods for PIK3R5 expression?

Interpreting conflicting results between different detection methods for PIK3R5 requires a systematic analytical approach:

• Method-specific limitations assessment:

  Detection Method	Common Limitations	Potential PIK3R5-Specific Issues
  Western Blot	Denatures proteins, loses spatial information	Observed molecular weight may vary from predicted 97.2 kDa due to post-translational modifications
  IHC/IF	Potential cross-reactivity, epitope masking	Mouse-derived antibodies may require special blocking when used on mouse tissues
  qPCR	Measures mRNA not protein, misses post-transcriptional regulation	Multiple transcript variants may not be detected by all primer sets
  Flow Cytometry	Cell permeabilization may affect epitope recognition	Fixation can alter PIK3R5 detection depending on antibody epitope

• Epitope availability analysis:

  • Different antibodies target different PIK3R5 regions (N-terminal, C-terminal, internal)

  • Method-dependent protein conformations may expose or conceal specific epitopes

  • Solution: Use multiple antibodies targeting different regions to build a comprehensive understanding

• Specificity verification:

  • Validate antibody specificity using overexpression systems as positive controls

  • Test detection in multiple cell lines with varying PIK3R5 expression (HepG2, HeLa, SVT2, A549, COS7, Jurkat, MDCK, PC12, MCF7)

  • Consider genetic approaches (siRNA knockdown, CRISPR knockout) to confirm signal specificity

• Resolution of conflicts through complementary approaches:

  • For WB vs. IHC discrepancies: Consider subcellular fractionation to determine if localized pools explain differences

  • For protein vs. mRNA discrepancies: Investigate post-transcriptional regulation through pulse-chase experiments

  • For differences between antibodies: Map the exact epitopes and consider epitope accessibility in different assays

• Biological context consideration:

  • PIK3R5 expression and localization may be stimulus-dependent or cell cycle-regulated

  • Confirm experimental conditions are truly comparable between methods

  • Document the physiological state of samples across all detection methods

When properly analyzed, seemingly conflicting results often reveal important biological insights about PIK3R5 regulation, localization, or post-translational modifications rather than technical artifacts.

How can PIK3R5 antibodies be utilized in cancer research and potential therapeutic target identification?

PIK3R5 antibodies offer multiple approaches for cancer research and therapeutic target identification:

• Expression profiling across cancer types:

  • Use immunohistochemistry with PIK3R5 antibodies to screen tissue microarrays across multiple cancer types

  • Compare expression levels in matched normal/tumor samples using Western blot quantification

  • Correlate PIK3R5 expression with clinical parameters and patient outcomes

• Signaling pathway analysis:

  • Employ phospho-specific antibodies alongside PIK3R5 detection to map activation states of downstream effectors

  • Use PIK3R5 antibodies in multiplexed immunofluorescence to visualize colocalization with other PI3K pathway components

  • Monitor PIK3R5 expression/localization changes in response to pathway inhibitors

• Mechanistic studies:

  • Immunoprecipitate PIK3R5 to identify novel binding partners in cancer cells versus normal cells

  • Use antibodies to track PIK3R5 subcellular localization changes during cancer progression

  • Investigate post-translational modifications of PIK3R5 in cancer contexts

• Therapeutic development applications:

  • Screen for compounds that disrupt critical PIK3R5 protein-protein interactions

  • Use antibodies to validate target engagement in drug development pipelines

  • Develop antibody-drug conjugates targeting cancer cells with aberrant PIK3R5 expression

• Biomarker development:

  • Validate PIK3R5 as a diagnostic or prognostic biomarker using antibody-based assays

  • Develop standardized immunohistochemical scoring systems for PIK3R5 in different cancer contexts

  • Correlate PIK3R5 expression with response to PI3K pathway inhibitors

• Resistance mechanism investigation:

  • Monitor changes in PIK3R5 expression/localization in models of acquired resistance

  • Use antibodies to identify compensatory signaling networks that emerge following treatment

These applications leverage the specificity of PIK3R5 antibodies to advance understanding of cancer biology and identify novel therapeutic approaches targeting the PI3K pathway.

What are the recommended protocols for using PIK3R5 antibodies in multiplexed immunofluorescence studies?

Implementing successful multiplexed immunofluorescence with PIK3R5 antibodies requires careful optimization:

• Antibody panel design:

  • Select PIK3R5 antibodies from different host species than other target antibodies in your panel

  • For mouse-derived PIK3R5 antibodies (like OTI4G9), pair with rabbit, goat, or rat antibodies for other targets

  • Verify that secondary antibodies have minimal cross-reactivity

• Sequential staining protocol:

  • For challenging combinations, implement sequential staining with stripping or blocking steps

  • Order of antibody application: begin with lowest abundance target (often PIK3R5) to maximize detection

  • Consider using directly conjugated primary antibodies to simplify multiplexing

• Sample preparation optimization:

  • Select fixation method that preserves all target epitopes (4% PFA is generally suitable)

  • Optimize antigen retrieval carefully - different epitopes may require different conditions

  • For tissues, reduce autofluorescence using treatments like Sudan Black B or commercial reagents

• Signal separation strategies:

  • Use spectral imaging and unmixing for fluorophores with overlapping spectra

  • Design panels with maximally separated excitation/emission profiles

  • If using tyramide signal amplification, perform sequential TSA with microwave treatment between rounds

• Validation controls:

  • Include single-color controls for accurate compensation/unmixing

  • Prepare absorption controls by pre-incubating antibodies with immunizing peptides

  • Compare multiplexed staining patterns with single-staining results to identify interference

• PIK3R5-specific considerations:

  • Start with recommended 1:100 dilution for immunofluorescence applications

  • For co-localization studies with PI3K pathway components, carefully validate antibody compatibility

  • When using mouse PIK3R5 antibodies on mouse tissues, implement Mouse-On-Mouse blocking reagents

• Image acquisition and analysis:

  • Acquire z-stacks to capture complete spatial information

  • Use appropriate controls for colocalization analysis (Pearson's coefficient, Manders' overlap)

  • Consider advanced analysis tools for quantifying spatial relationships between PIK3R5 and other proteins

Following these protocols enables robust multiplexed detection of PIK3R5 alongside other proteins of interest for comprehensive pathway analysis.

How does PIK3R5 expression and function vary across different cell types and experimental models?

PIK3R5 exhibits significant variation in expression and function across cell types and experimental models:

• Cell line expression patterns:

  • Western blot analysis reveals detectable PIK3R5 expression across diverse cell lines including HepG2, HeLa, SVT2, A549, COS7, Jurkat, MDCK, PC12, and MCF7

  • Expression levels vary considerably, with immune cells (e.g., Jurkat) often showing higher baseline expression

  • Species conservation allows detection across human, mouse, rat, canine, and monkey cell lines

• Tissue-specific expression profiles:

  • Highest expression observed in immune tissues and cells, consistent with PIK3R5's role in immune signaling

  • Brain tissues show distinct regional variation in PIK3R5 expression

  • Expression can be induced in some tissues/cells following specific stimuli (particularly inflammatory signals)

• Subcellular localization differences:

  • Primarily cytoplasmic in resting cells, but can show membrane translocation upon activation

  • Nuclear localization observed in some cell types, suggesting non-canonical functions

  • Localization patterns can be visualized using immunofluorescence at 1:100 antibody dilution

• Functional variation by cell type:

  Cell Type	Primary PIK3R5 Function	Detection Notes
  Immune cells	Regulates chemotaxis and inflammatory responses	Higher baseline expression
  Endothelial cells	Mediates angiogenic responses	Expression increases under hypoxic conditions
  Neurons	Involved in neurotrophin signaling	Region-specific expression patterns
  Cancer cells	Often dysregulated, promoting proliferation and survival	Expression correlates with aggressive phenotypes in some cancers

• Model system considerations:

  • Knockout models reveal more severe phenotypes in immune and cardiovascular systems

  • Overexpression systems (e.g., HEK293T transfection models) useful for antibody validation

  • Patient-derived xenografts may maintain PIK3R5 expression patterns of original tumors

• Experimental induction factors:

  • Expression and activation responsive to growth factors, cytokines, and cellular stress

  • Post-translational modifications (particularly phosphorylation) regulate function independently of expression level

  • Interaction with PIK3CG is context-dependent and can be monitored with specific antibodies

Understanding these variations is essential for proper experimental design and interpretation of results when studying PIK3R5 across different biological contexts.

How do post-translational modifications affect PIK3R5 detection with antibodies?

Post-translational modifications (PTMs) significantly impact PIK3R5 detection with antibodies through several mechanisms:

• Epitope masking effects:

  • Phosphorylation events near antibody epitopes can block antibody binding

  • Most commercial PIK3R5 antibodies target specific regions (N-terminal, C-terminal, or internal domains)

  • Researchers should note that the observed molecular weight may differ from the theoretical 97.2 kDa due to PTMs

• Modification-specific detection challenges:

  • Phosphorylation: Major regulatory mechanism for PIK3R5 function

  • Ubiquitination: Can affect protein turnover and detection sensitivity

  • Glycosylation: May alter antibody accessibility to protein backbone epitopes

• Methodological considerations by technique:

  Technique	PTM Impact	Mitigation Strategy
  Western Blot	Altered migration patterns	Include phosphatase/deglycosylase treated controls
  IP	Modified epitopes may affect pull-down efficiency	Use multiple antibodies targeting different regions
  IHC/IF	Fixation can preserve or destroy PTMs	Optimize fixation protocols for PTM preservation

• Stimulus-dependent modification patterns:

  • Activation of PI3K signaling induces specific phosphorylation patterns

  • Experimental treatments may alter PTM status without changing expression

  • Time-course studies are recommended to capture dynamic modification changes

• Cell type-specific modification profiles:

  • Different cell types exhibit distinct PTM patterns on PIK3R5

  • The same antibody may show variable detection efficiency across cell types

  • Validation across multiple cell types is recommended for new applications

• Specialized detection approaches:

  • For comprehensive PTM analysis, consider using antibodies targeting specific PIK3R5 modifications

  • Phospho-specific antibodies can complement total PIK3R5 detection

  • Mass spectrometry following immunoprecipitation with PIK3R5 antibodies can identify novel modifications

Researchers should document experimental conditions that may affect PIK3R5 modification status and consider how these modifications might impact antibody detection when interpreting results across different experimental contexts.

What cross-reactivity issues should researchers be aware of when using PIK3R5 antibodies?

Researchers should be vigilant about several cross-reactivity issues when working with PIK3R5 antibodies:

• Species cross-reactivity considerations:

  • Most commercial PIK3R5 antibodies show cross-reactivity across multiple species including human, mouse, rat, dog, and monkey

  • Sequence homology can lead to unintended reactivity in non-validated species

  • Validate antibodies in your specific species of interest even if listed as reactive

• Mouse-on-mouse reactivity issues:

  • When using mouse-derived antibodies (like OTI4G9) on mouse tissues, high background is common

  • Special blocking reagents are recommended (e.g., those referenced as PK-2200-NB and MP-2400-NB)

  • Consider alternative host species antibodies when working with mouse samples

• PI3K family cross-reactivity:

  • PIK3R5 shares structural domains with other PI3K regulatory subunits

  • Antibodies targeting conserved regions may cross-react with PIK3R1, PIK3R2, or PIK3R3

  • Validation using overexpression systems can help confirm specificity

• Isoform-specific detection challenges:

  • Multiple isoforms or splice variants of PIK3R5 may exist

  • Antibodies may preferentially detect certain isoforms depending on epitope location

  • Document which isoforms are detected by your specific antibody

• Application-specific cross-reactivity:

  Application	Common Cross-Reactivity Issues	Mitigation Strategy
  Western Blot	Secondary bands at unexpected molecular weights	Include positive controls with known PIK3R5 expression
  IHC/IF	Non-specific tissue binding	Include absorption controls with immunizing peptide
  IP	Co-precipitation of binding partners	Use stringent washing and validate with reciprocal IP

• Commercial antibody validation:

  • Most commercial antibodies are validated on a limited set of samples

  • The OTI4G9 clone has been validated across multiple cell lines (HepG2, HeLa, SVT2, A549, COS7, Jurkat, MDCK, PC12, MCF7)

  • Epitope-specific antibodies (e.g., targeting AA 763-792, C-Term) may have different cross-reactivity profiles

• Technical remediation approaches:

  • Preabsorption with immunizing peptides can confirm specificity

  • Side-by-side comparison of multiple PIK3R5 antibodies targeting different epitopes

  • Genetic approaches (siRNA, CRISPR) provide definitive specificity controls

Thorough validation of PIK3R5 antibodies in your specific experimental system is essential to identify and address potential cross-reactivity issues before conducting critical experiments.

What are the latest advances in using PIK3R5 antibodies for single-cell analysis techniques?

Recent advances in using PIK3R5 antibodies for single-cell analysis represent significant methodological breakthroughs:

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated PIK3R5 antibodies enable high-dimensional analysis alongside dozens of other markers

  • Palladium or rare earth metal conjugates provide minimal signal overlap

  • Allows correlation of PIK3R5 expression with cell type, activation state, and other signaling molecules at single-cell resolution

  • Most effective with antibodies validated for flow cytometry applications

• Single-cell imaging mass spectrometry:

  • Antibodies conjugated to mass tags enable spatial analysis of PIK3R5 in tissue contexts

  • Multiplexed ion beam imaging (MIBI) and multiplexed immunofluorescence techniques allow simultaneous detection of PIK3R5 and 40+ other proteins

  • Preserves tissue architecture while providing single-cell resolution

• Microfluidic approaches:

  • Antibody-based capture of PIK3R5-expressing cells in microfluidic devices

  • Single-cell western blotting techniques using PIK3R5 antibodies at 1:500-2000 dilution

  • Combination with single-cell RNA-seq allows correlation of protein and transcript levels

• In situ protein analysis:

  • Proximity ligation assays (PLA) with PIK3R5 antibodies detect protein-protein interactions in single cells

  • Highly multiplexed immunofluorescence with signal amplification improves detection of low-abundance PIK3R5

  • Cyclic immunofluorescence methods enable detection of PIK3R5 alongside 50+ other proteins in the same sample

• Live-cell applications:

  • Membrane-permeable PIK3R5 antibody derivatives for intracellular tracking

  • Nanobody-based detection systems with reduced steric hindrance

  • Integration with optogenetic approaches for simultaneous perturbation and detection

• Computational analysis integration:

  • Machine learning algorithms to identify PIK3R5 expression patterns in heterogeneous cell populations

  • Trajectory analysis to map PIK3R5 dynamics during cellular processes

  • Spatial statistics to quantify PIK3R5 colocalization with pathway components at subcellular resolution

• Validation approaches for single-cell techniques:

  • Correlation with bulk measurements to confirm signal specificity

  • Genetic manipulation (CRISPR knockout) controls for antibody specificity

  • Spike-in controls with cells expressing known PIK3R5 levels

These advances are transforming our understanding of PIK3R5 biology by revealing cell-to-cell variability and context-dependent functions that were previously obscured in bulk analyses.