PIK3R2 Antibody, FITC conjugated

What is PIK3R2 and why is it significant in cellular signaling research?

PIK3R2, also known as phosphatidylinositol 3-kinase regulatory subunit beta (p85β), is a regulatory subunit of PI3K that plays a critical role in the PI3K/AKT signaling pathway. This pathway is fundamental in regulating cellular processes including cell growth, survival, proliferation, and motility. PIK3R2 functions primarily in the cytoplasm, Golgi apparatus, and nucleus . Its importance in research stems from its involvement in various physiological processes and pathological conditions, including cancer and metabolic disorders.

The regulatory subunits of PI3K, including PIK3R2, are responsible for mediating the interaction between PI3K and receptor tyrosine kinases, thereby controlling PI3K activation and subsequent signaling cascades. Understanding PIK3R2 expression and localization provides insights into PI3K pathway regulation and its role in disease mechanisms.

What are the technical advantages of FITC-conjugated antibodies for PIK3R2 detection?

FITC (Fluorescein isothiocyanate) conjugated antibodies offer several technical advantages for PIK3R2 detection in research applications:

• Direct detection without secondary antibodies, streamlining protocols and reducing background noise

• Compatibility with multiple fluorescence-based techniques including immunofluorescence, flow cytometry, and confocal microscopy

• Strong fluorescence emission at 520 nm (green spectrum) when excited at 495 nm

• Ability to be combined with other fluorophores in multi-color imaging experiments

• Particularly useful for analysis of subcellular localization patterns of PIK3R2

When properly stored (typically at -20°C in aliquots to avoid repeated freeze-thaw cycles), FITC-conjugated antibodies maintain their fluorescence properties and specificity for extended periods .

What experimental applications are most suitable for PIK3R2 antibody detection?

Based on validated research protocols, PIK3R2 antibodies are suitable for multiple experimental applications:

The compatibility of PIK3R2 antibodies across multiple applications makes them versatile tools for comprehensive protein analysis in both basic and translational research settings.

How can researchers optimize PIK3R2 antibody staining in different tissue types?

Optimization of PIK3R2 antibody staining across different tissue types requires systematic adjustment of several parameters:

For paraffin-embedded tissues:

• Perform heat-induced epitope retrieval using 10mM Tris with 1mM EDTA, pH 9.0, for 45 minutes at 95°C followed by 20 minutes cooling at room temperature

• Test antibody concentration range (typically 1-2 μg/ml is effective) with 30-minute incubation at room temperature

• Include positive control tissues (spleen shows consistent PIK3R2 expression)

• Implement tissue-specific blocking strategies to minimize background

For frozen sections and cultured cells:

• Fixation method significantly impacts staining quality (4% paraformaldehyde generally preserves PIK3R2 epitopes while maintaining cellular architecture)

• Permeabilization requires optimization (0.1-0.3% Triton X-100 for membrane permeabilization)

• Extended primary antibody incubation (overnight at 4°C) often yields superior signal-to-noise ratio compared to shorter incubations

• Validate specificity using PIK3R2 knockdown or knockout samples when available

What experimental controls should be implemented when working with FITC-conjugated PIK3R2 antibodies?

Rigorous experimental controls are essential for generating reliable and interpretable data with FITC-conjugated PIK3R2 antibodies:

Positive Controls:

• Cell lines with validated PIK3R2 expression (based on literature or western blot validation)

• Tissues with known PIK3R2 expression patterns (human spleen shows consistent staining)

Negative Controls:

• Isotype controls matching the primary antibody host and isotype (Mouse IgG2b for monoclonal antibodies)

• Secondary antibody-only controls to assess non-specific binding

• Antigen competition assays using recombinant PIK3R2 protein

Fluorescence Controls:

• Unstained samples to establish autofluorescence baseline

• Single-color controls when performing multicolor experiments

• FITC fluorescence quenching controls to distinguish genuine signal reduction from experimental artifacts

Implementing these controls enables accurate interpretation of experimental results and facilitates troubleshooting when unexpected staining patterns occur.

How can researchers troubleshoot weak or non-specific staining with PIK3R2 antibodies?

When encountering staining issues with PIK3R2 antibodies, systematic troubleshooting approaches should be employed:

For Weak Staining:

• Increase antibody concentration incrementally (starting from 1 μg/ml up to 5 μg/ml)

• Extend incubation time (from 30 minutes to overnight at 4°C)

• Optimize antigen retrieval conditions (test different buffers and pH levels)

• Ensure proper storage of antibody (repeated freeze-thaw cycles significantly reduce activity)

• Verify sample preparation methods preserve PIK3R2 epitopes

For Non-specific Staining:

• Implement more stringent blocking protocols (5% BSA or serum from the same species as the secondary antibody)

• Reduce antibody concentration

• Include additional washing steps with 0.1% Tween-20

• Validate antibody specificity using western blot prior to immunostaining

• Consider alternative PIK3R2 antibody clones if persistent issues occur

For FITC-Specific Issues:

• Minimize exposure to light throughout the staining procedure

• Use anti-fade mounting media to prevent photobleaching

• Store slides in the dark at 4°C and image promptly

• Consider signal amplification methods if FITC signal is consistently weak

What is the optimal sample preparation protocol for PIK3R2 immunofluorescence studies?

Optimal sample preparation for PIK3R2 immunofluorescence studies involves several critical steps:

For Cultured Cells:

• Grow cells on glass coverslips to 70-80% confluence

• Wash gently with PBS (pH 7.4) to remove media components

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum (from secondary antibody species) with 1% BSA for 1 hour

• Incubate with FITC-conjugated PIK3R2 antibody (typically 1-5 μg/ml) for 1-2 hours at room temperature or overnight at 4°C

• Wash extensively with PBS containing 0.05% Tween-20

• Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

• Mount with anti-fade media

For Tissue Sections:

• Fix tissues promptly in 10% neutral buffered formalin

• Process and embed in paraffin following standard protocols

• Section at 4-6 μm thickness

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval using 10mM Tris with 1mM EDTA (pH 9.0) for 45 minutes at 95°C

• Cool sections at room temperature for 20 minutes

• Block endogenous peroxidase (if applicable) and non-specific binding sites

• Apply FITC-conjugated PIK3R2 antibody at 1-2 μg/ml concentration

• Proceed with washing and counterstaining as for cultured cells

How should researchers determine the optimal concentration of FITC-conjugated PIK3R2 antibody?

Determining the optimal antibody concentration requires systematic titration:

• Initial Concentration Range Testing:

  • Prepare a concentration series (typically 0.5, 1, 2, 5, and 10 μg/ml)

  • Maintain all other experimental conditions constant

  • Process samples in parallel to enable direct comparison

• Signal-to-Noise Evaluation:

  • Quantitatively assess signal intensity in regions of expected PIK3R2 expression

  • Measure background fluorescence in regions expected to lack PIK3R2

  • Calculate signal-to-noise ratios for each concentration

• Validation Across Sample Types:

  • Test optimized concentration across different tissue or cell types

  • Verify consistent performance in biological replicates

  • Assess whether optimization parameters transfer between different experimental platforms

• Standard Curve Generation:

  • For quantitative applications, establish a standard curve using recombinant PIK3R2 protein

  • Determine the linear detection range of the antibody

The optimal concentration typically provides maximum specific signal with minimal background staining, usually in the range of 1-2 μg/ml for immunohistochemical applications .

What are the recommended approaches for multiplex staining involving FITC-conjugated PIK3R2 antibody?

Multiplex staining with FITC-conjugated PIK3R2 antibody requires careful experimental design:

Fluorophore Selection:

• Choose complementary fluorophores with minimal spectral overlap (e.g., FITC paired with Cy3, Cy5, or APC)

• Consider the excitation/emission capabilities of available microscopy equipment

• Account for relative signal strengths of each target protein

Sequential Staining Protocol:

• Begin with heat-induced epitope retrieval suitable for all target antigens

• Block non-specific binding sites thoroughly (5% normal serum with 1% BSA)

• Apply the first primary antibody (non-FITC conjugated)

• Detect with appropriate secondary antibody

• Apply additional blocking step to prevent cross-reactivity

• Apply FITC-conjugated PIK3R2 antibody (typically last in the sequence to minimize photobleaching)

• Counterstain nuclei with DAPI or similar nuclear stain

• Mount with anti-fade mounting medium

Controls for Multiplex Staining:

• Single-stained samples for each antibody to establish spectral profiles

• Absorption controls to confirm absence of spectral bleed-through

• Biological controls where co-expression patterns are well-established

When studying PIK3R2 in context with its interaction partners or downstream effectors, multiplex staining provides valuable insights into spatial relationships and co-localization patterns within cellular compartments.

How can PIK3R2 antibodies be used to investigate PI3K pathway dysregulation in disease models?

PIK3R2 antibodies serve as valuable tools for investigating PI3K pathway dysregulation:

• Expression Level Analysis:

  • Quantitative assessment of PIK3R2 protein expression in normal versus diseased tissues

  • Correlation of expression levels with disease progression or patient outcomes

  • Detection of altered PIK3R2/p110 catalytic subunit ratios that may impact signaling dynamics

• Subcellular Localization Studies:

  • Tracking altered PIK3R2 distribution between cytoplasm, membrane, and nucleus

  • Identification of abnormal PIK3R2 recruitment to signaling complexes

  • Visualization of compartment-specific PIK3R2 functions using FITC-conjugated antibodies

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation followed by western blot to identify PIK3R2 binding partners

  • Proximity ligation assays to visualize protein interactions in situ

  • Assessment of how disease-associated mutations affect interaction networks

• Functional Pathway Analysis:

  • Correlation of PIK3R2 expression/localization with phosphorylation of downstream targets (e.g., AKT)

  • Assessment of PIK3R2 status following therapeutic pathway inhibition

  • Evaluation of compensatory mechanisms involving PIK3R2 during targeted therapy

What methodological considerations apply when using PIK3R2 antibodies in phosphorylation state detection?

Detecting phosphorylated forms of PIK3R2 requires specialized approaches:

• Sample Preparation:

  • Rapid sample processing to preserve phosphorylation status

  • Inclusion of phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers

  • Maintenance of cold temperatures throughout processing

• Antibody Selection:

  • Use of phospho-specific PIK3R2 antibodies when available

  • Validation of phospho-specificity using phosphatase treatment controls

  • Comparison with total PIK3R2 detection using separate antibodies

• Signal Enhancement Strategies:

  • Tyramide signal amplification for weak phospho-specific signals

  • Optimized antigen retrieval specific for phospho-epitopes

  • Extended antibody incubation times at 4°C to maximize sensitivity

• Quantitative Analysis:

  • Ratiometric analysis of phosphorylated to total PIK3R2

  • Calibration with phosphorylation standards when available

  • Statistical validation across multiple biological replicates

How do researchers interpret conflicting data when using different PIK3R2 antibody clones?

When faced with conflicting results from different PIK3R2 antibody clones, researchers should implement a systematic investigation:

• Epitope Mapping Analysis:

  • Determine the specific epitope regions recognized by each antibody

  • Assess whether epitopes might be masked by protein interactions or conformational changes

  • Consider whether post-translational modifications might affect epitope accessibility

• Validation Through Orthogonal Methods:

  • Confirm PIK3R2 expression using mRNA detection methods

  • Implement genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • Use mass spectrometry to confirm protein identity in immunoprecipitation studies

• Cross-Platform Comparison:

  • Test antibodies across multiple applications (WB, IHC, IF)

  • Determine whether discrepancies are application-specific

  • Evaluate whether fixation or sample preparation methods differentially affect epitope detection

• Standardization Approach:

  • Select antibodies validated against recombinant PIK3R2 protein

  • Prioritize monoclonal antibodies with well-characterized epitopes

  • Establish a consistent protocol across research groups to minimize technical variability

A comprehensive table documenting the performance characteristics of different antibody clones across applications can help identify the most reliable reagents for specific experimental questions.

What are the technical challenges in distinguishing PIK3R2 from other PI3K regulatory subunits?

Distinguishing PIK3R2 (p85β) from other PI3K regulatory subunits, particularly PIK3R1 (p85α), presents several technical challenges:

• Sequence Homology Management:

  • PIK3R1 and PIK3R2 share significant sequence homology

  • Verification of antibody specificity through western blot analysis of recombinant proteins

  • Implementation of peptide competition assays to confirm epitope specificity

• Cross-Reactivity Assessment:

  • Testing antibodies against cells with genetic deletion of specific regulatory subunits

  • Comparative analysis using multiple antibodies targeting different epitopes

  • Specificity validation using recombinant expression systems

• Isoform-Specific Detection Strategies:

  • Use of antibodies raised against the most divergent regions between isoforms

  • Implementation of isoform-specific PCR as complementary validation

  • Development of selective immunoprecipitation protocols followed by mass spectrometry

• Quantitative Discrimination:

  • Careful calibration using purified proteins of known concentration

  • Establishment of standard curves for each isoform

  • Statistical methods to account for potential cross-reactivity

The immunogen used for antibody production is critical - antibodies raised against amino acids 520-720 of human PIK3R2 show good specificity .

How can researchers effectively store and maintain FITC-conjugated PIK3R2 antibodies to ensure long-term stability?

Maintaining the stability and performance of FITC-conjugated PIK3R2 antibodies requires attention to several storage parameters:

• Temperature Management:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing small single-use aliquots

  • Allow antibodies to equilibrate to room temperature before opening tubes

• Light Protection:

  • Store in amber or opaque tubes to protect from light exposure

  • Minimize exposure to light during experimental procedures

  • Consider working under reduced ambient lighting when handling

• Buffer Optimization:

  • Storage in buffers containing stabilizing proteins (0.1-1% BSA)

  • Inclusion of preservatives such as 0.05% sodium azide

  • Consideration of specialized stabilizing buffers for prolonged storage

• Quality Control Measures:

  • Periodic testing of antibody performance over time

  • Documentation of fluorescence intensity compared to initial levels

  • Implementation of standardized positive controls to track potential degradation

Stability Monitoring Protocol:

• Test aliquots at defined intervals (0, 3, 6, 12 months)

• Compare fluorescence intensity and specificity to initial performance

• Document any changes in background or non-specific binding

What quantitative methods can reliably measure PIK3R2 expression levels using antibody-based techniques?

Several quantitative approaches can be employed to measure PIK3R2 expression levels:

• Western Blot Densitometry:

  • Careful sample loading normalization using housekeeping proteins

  • Use of standard curves with recombinant PIK3R2 protein

  • Digital image analysis with appropriate software (ImageJ, Bio-Rad Image Lab)

  • Statistical analysis across multiple biological replicates

• Quantitative Immunofluorescence:

  • Calibration using fluorescence standards

  • Confocal microscopy with standardized acquisition parameters

  • Automated image analysis to measure integrated density values

  • Single-cell analysis to assess population heterogeneity

• Flow Cytometry:

  • Calibration using fluorescent beads of known intensity

  • Mean fluorescence intensity (MFI) quantification

  • Conversion of arbitrary units to absolute molecule numbers

  • Comparison against isotype controls

• ELISA-Based Methods:

  • Development of sandwich ELISA using capture and detection antibodies

  • Generation of standard curves with recombinant PIK3R2

  • Measurement of PIK3R2 in cell or tissue lysates

  • Statistical validation of assay precision and reproducibility

Recommended Approach for Tissue Analysis:
Quantitative immunohistochemistry using digital pathology platforms with:

• Automated tissue segmentation

• Standardized staining protocols

• Internal calibration standards

• Multi-parameter analysis correlating PIK3R2 with other biomarkers