PIK3R2 Antibody, Biotin conjugated

What is PIK3R2 and what cellular functions does it mediate?

PIK3R2, also known as phosphoinositide-3-kinase regulatory subunit 2 (beta) or p85β, is a critical regulatory component of Class IA PI3Ks. It functions primarily as an adaptor protein that stabilizes and inhibits the p110 catalytic subunit in basal conditions. Upon receptor activation, PIK3R2 mediates the recruitment of the PI3K complex to activated receptors, relieving inhibition of the catalytic subunit.

The protein is involved in multiple signaling pathways including cell growth, proliferation, differentiation, motility, and intracellular trafficking. PIK3R2 is part of the PI3K family that phosphorylates phosphoinositides on the 3-hydroxyl group of the inositol ring . It plays important roles in:

• Modulation of extracellular signals

• E-cadherin-mediated cell-cell adhesion

• Maintenance of epithelial structural integrity

• Assembly of adherens junctions

How do biotin-conjugated antibodies enhance detection strategies?

Biotin-conjugated antibodies provide significant advantages in complex experimental systems due to the following characteristics:

• High-affinity interactions: The biotin-streptavidin/avidin system features one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), ensuring stable detection complexes

• Signal amplification: Each biotin molecule can bind multiple streptavidin molecules, which allows for significant signal enhancement

• Compatibility with multiple detection systems: Biotin-conjugated antibodies can be coupled with various detection molecules including fluorophores, enzymes, or gold particles through streptavidin conjugates

• Reduced background: The system often produces cleaner results with less non-specific binding in applications like immunohistochemistry

In research contexts, biotin-conjugated PIK3R2 antibodies enable sensitive detection in applications such as ELISA (recommended dilution 1:1000), Western blotting (recommended dilution 1:50-100), and immunohistochemistry (recommended dilution 1:10-50) .

What are the optimal storage conditions for maintaining PIK3R2 antibody activity?

Proper storage is critical for maintaining antibody performance. Based on manufacturer specifications, biotin-conjugated PIK3R2 antibodies should be stored as follows:

• Temperature: Store at -20°C for up to 12 months

• Buffer composition: Antibodies are typically supplied in aqueous buffered solutions containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

• Aliquoting: Divide the antibody into small aliquots before freezing to avoid repeated freeze-thaw cycles

• Thawing protocol: Thaw on ice and mix gently by inversion; avoid vortexing which can damage the antibody structure

The inclusion of glycerol (50%) in the storage buffer serves as a cryoprotectant to prevent damage during freeze-thaw cycles, while the BSA helps stabilize the antibody protein and prevent adsorption to storage containers.

What are the validated applications for PIK3R2 antibodies?

PIK3R2 antibodies have been validated for multiple research techniques, with application-specific dilution recommendations:

The antibody has been validated with human samples, with immunohistochemical analysis showing specific staining in human spleen tissue . When using these antibodies, proper validation in your specific experimental system is recommended.

How can researchers validate specificity when working with PIK3R2 antibodies?

Rigorous validation of PIK3R2 antibodies is essential for reliable experimental outcomes. A comprehensive validation strategy should include:

1. Positive and negative controls:

• Positive controls: Use tissues or cell lines known to express high levels of PIK3R2 (e.g., human spleen tissue)

• Negative controls:

  • Isotype controls such as Mouse IgG2b [PLRV219] (A86740) or Mouse IgG2b [MPC-11] (A86389)

  • Tissues/cells with genetically silenced PIK3R2 expression (siRNA or CRISPR-edited)

2. Peptide competition assay:

• Pre-incubate the antibody with purified PIK3R2 protein or immunizing peptide

• Compare staining patterns between blocked and unblocked antibody

• Specific signals should be diminished or eliminated in the blocked sample

3. Cross-reactivity assessment:

• Test reactivity against related PI3K family members, particularly PIK3R1 (p85α)

• Confirm specificity through immunoprecipitation followed by mass spectrometry

4. Multiple antibody validation:

• Use multiple antibodies targeting different epitopes of PIK3R2

• Consistent results across different antibodies increase confidence in specificity

For phospho-specific PIK3R2 antibodies (e.g., those targeting phosphorylated residues like Thr1024 in the related PIK3CG), additional validation using phosphatase treatment of samples should be performed .

What are the critical factors for optimizing multiplex immunoassays with biotin-conjugated PIK3R2 antibodies?

Developing effective multiplex assays with biotin-conjugated PIK3R2 antibodies requires careful consideration of several technical parameters:

1. Blocking strategy optimization:

• Use biotin-free blocking agents to prevent streptavidin binding to endogenous biotin

• Consider avidin/streptavidin pre-blocking when working with tissues containing high endogenous biotin (e.g., liver, kidney)

2. Antibody panel design:

• Select complementary antibodies with compatible isotypes when using multiple primary antibodies

• When using rabbit polyclonal PIK3R2 antibodies , ensure other antibodies in the panel don't cross-react with rabbit immunoglobulins

3. Signal development sequence:

• In multi-color immunofluorescence:

  • Apply fluorophore-conjugated streptavidin after all other secondary antibodies

  • Use streptavidin conjugated to fluorophores with minimal spectral overlap with other channels

4. Titration optimization:

• Conduct antibody titration experiments to determine optimal concentration

• For immunohistochemistry applications, begin with dilutions of 1:10-50

• For Western blotting, test dilutions ranging from 1:50-100

5. Signal amplification methods:

• Tyramide signal amplification (TSA) can be combined with biotin-streptavidin systems

• When using amplification methods, reduce primary antibody concentration to maintain specificity

How do different PIK3R2 antibody clones compare in terms of epitope recognition and performance?

Different PIK3R2 antibody clones recognize distinct epitopes and demonstrate varying performance characteristics across applications:

When selecting between these options, researchers should consider:

• Application requirements: Some antibody clones perform better in specific applications

• Sample type: Different clones may have varying efficacy in different tissues or preparations

• Detection system compatibility: Consider downstream detection requirements

• Reproducibility needs: Monoclonal antibodies generally provide more consistent results across experiments

What strategies can address cross-reactivity challenges with PIK3 family antibodies?

The PIK3 family contains multiple isoforms with high sequence homology, presenting challenges for specific detection. To manage cross-reactivity:

1. Epitope selection considerations:

• Choose antibodies targeting unique regions of PIK3R2

• Some commercially available antibodies are derived from synthetic peptides specifically selected to maximize isoform specificity

2. Validation in knockout/knockdown systems:

• Confirm specificity using PIK3R2 knockout or knockdown systems

• Compare staining patterns between wild-type and PIK3R2-deficient samples

3. Sequential immunoprecipitation approach:

• First deplete samples of highly homologous proteins (e.g., PIK3R1) using specific antibodies

• Then probe for PIK3R2 in the depleted sample

4. Computational prediction and experimental verification:

• Use bioinformatic tools to predict cross-reactivity based on epitope sequence homology

• Experimentally verify predictions using purified recombinant proteins

5. Isoform-specific detection strategies:

• When available, use phosphorylation-specific antibodies that target modifications unique to PIK3R2

• Consider RNA-based methods (RT-PCR, RNA-seq) as complementary approaches to confirm protein identification

What troubleshooting approaches are effective for inconsistent PIK3R2 antibody performance?

When encountering inconsistent results with PIK3R2 antibodies, consider this systematic troubleshooting approach:

1. Sample preparation optimization:

• Fixation impact: Different fixation methods can affect epitope accessibility

  • For formalin-fixed tissues, optimize antigen retrieval (test heat-induced vs. enzymatic methods)

  • For phospho-specific detection, use phosphatase inhibitors throughout sample preparation

2. Detection system considerations:

• Biotin amplification issues:

  • Test for endogenous biotin interference using streptavidin-only controls

  • If background is high, consider biotin-blocking steps or alternative detection systems

3. Protocol modifications for challenging samples:

• High-background tissues:

  • Increase blocking duration (2-24 hours)

  • Use specialized blocking reagents containing both proteins and mild detergents

  • Consider longer washing steps with gentle agitation

4. Antibody-specific parameters:

• Concentration optimization:

  • Create a titration series for each new lot of antibody

  • For Western blotting, test dilutions from 1:50-5000

  • For ELISA applications, begin with 1:500-1000

5. Signal-to-noise enhancement:

• Temperature effects:

  • Try primary antibody incubation at different temperatures (4°C, room temperature)

  • Extended incubation at 4°C (overnight) can improve specific binding while reducing background

6. Lot-to-lot variation management:

• Validation benchmark:

  • Maintain a reference sample with known reactivity

  • Test each new antibody lot against the reference before use in critical experiments

Future directions in PIK3R2 antibody research applications

The development and application of PIK3R2 antibodies continues to evolve, with several emerging areas of interest:

• Single-cell applications: Advances in highly sensitive detection systems are enabling PIK3R2 detection at the single-cell level

• Multiplex imaging technologies: Integration of biotin-conjugated antibodies into highly multiplexed imaging platforms like cyclic immunofluorescence and mass cytometry

• Therapeutic monitoring: Using PIK3R2 antibodies to monitor pathway activity in clinical trials of PI3K pathway inhibitors

• Structural biology applications: Conformation-specific antibodies that can distinguish active vs. inactive states of the PI3K complex