PIK3AP1 Antibody, FITC conjugated

What is PIK3AP1 and what cellular functions does it serve?

PIK3AP1, also known as B-cell adapter for phosphoinositide 3-kinase (BCAP), is a signaling adapter protein that plays crucial roles in immune cell signaling. It contributes significantly to B-cell development by linking B-cell receptor (BCR) signaling to the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway . PIK3AP1 provides a docking site for the PI3K subunit PIK3R1, which effectively couples BCR and PI3K activation. Additionally, it connects Toll-like receptor (TLR) signaling to PI3K activation, a process that helps prevent excessive inflammatory cytokine production. Research has also demonstrated PIK3AP1's involvement in the activation of PI3K in natural killer cells and its potential role in mature B-cell survival through activation of REL transcription factor .

What signaling pathways involve PIK3AP1?

PIK3AP1 participates in several critical signaling cascades within immune cells. Primarily, it functions within the PI3K-Akt pathway, serving as a bridge between B-cell receptor (BCR) associated kinases and PI3K signaling components . During B-cell activation, tyrosine kinases including Syk, Btk, or Lyn phosphorylate PIK3AP1, creating binding sites for the p85 regulatory subunit of PI3K . Additionally, PIK3AP1 links Toll-like receptor (TLR) signaling to PI3K activation, thereby modulating inflammatory responses . The protein also participates in natural killer cell signaling networks and may influence B-cell survival mechanisms through REL activation . Recent research indicates that the PI3K-Akt pathway plays important roles during viral infections by promoting cell survival and inhibiting apoptosis, suggesting PIK3AP1's potential involvement in anti-viral immunity .

What experimental applications are suitable for PIK3AP1 Antibody, FITC conjugated?

PIK3AP1 Antibody, FITC conjugated can be employed in multiple experimental applications for investigating B-cell signaling and immune responses. Technical specifications indicate its suitability for Western blotting (recommended dilution 1:300-5000), immunofluorescence on paraffin-embedded tissues (IF/IHC-P; dilution 1:50-200), immunofluorescence on frozen tissues (IF/IHC-F; dilution 1:50-200), and immunocytochemistry (IF/ICC; dilution 1:50-200) . The FITC conjugation enables direct visualization without requiring secondary antibodies, making it particularly valuable for multicolor flow cytometry analyses of immune cell populations and for direct immunofluorescence microscopy applications. When using this antibody for subcellular localization studies, researchers should note that PIK3AP1 exhibits both cytoplasmic and membrane localization patterns , requiring appropriate imaging techniques to distinguish between these compartments.

What are the recommended storage conditions for PIK3AP1 Antibody, FITC conjugated?

For optimal preservation of both antibody function and fluorophore activity, PIK3AP1 Antibody, FITC conjugated should be stored at -20°C or -80°C . The antibody is typically supplied in a protective buffer containing glycerol (50%) to prevent freeze-thaw damage. According to product specifications, the storage buffer typically contains "0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4" or alternatively "0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol" . To maintain antibody integrity, it is strongly recommended to aliquot the stock solution into multiple small volumes upon initial thawing to avoid repeated freeze-thaw cycles . During experimental procedures, the antibody should be kept on ice and protected from light exposure to prevent photobleaching of the FITC fluorophore, which can significantly reduce signal intensity during imaging or flow cytometry applications.

What cell types express PIK3AP1 at detectable levels?

PIK3AP1 expression has been documented in multiple tissues and cell lineages relevant to immunological research. According to published data, PIK3AP1 mRNA is present in mouse spleen, thymus, liver, lung, macrophage populations, and B cell lines . As its name suggests, PIK3AP1 shows prominent expression in B lymphocytes, consistent with its functional role in B-cell receptor signaling pathways. The literature also indicates significant expression in natural killer cells, where it participates in activation of PI3K signaling . More recent research has demonstrated expression in bone marrow-derived macrophages (BMDMs), which were utilized in studies examining PIK3AP1's role during viral infection . The Human Protein Atlas provides additional information regarding PIK3AP1 expression patterns across human tissues . This expression profile should guide researchers in selecting appropriate cellular models for studying PIK3AP1 functions and validating antibody specificity in experimental systems.

How can PIK3AP1 Antibody, FITC conjugated be used to study the interaction between B-cell receptor signaling and the PI3K-Akt pathway?

For investigating interactions between BCR signaling and the PI3K-Akt pathway using PIK3AP1 Antibody, FITC conjugated, researchers can implement several methodological approaches. Flow cytometry represents an effective technique for monitoring changes in PIK3AP1 expression levels and cellular localization following BCR stimulation with anti-IgM antibodies. The direct FITC conjugation facilitates visualization of PIK3AP1 redistribution within activated B cells. To examine protein-protein interactions, co-localization studies can be performed by combining PIK3AP1 Antibody, FITC with antibodies against BCR components or PI3K subunits labeled with spectrally distinct fluorophores. This approach is particularly valuable given that PIK3AP1 provides a documented docking site for the PI3K subunit PIK3R1, thereby coupling BCR and PI3K activation .

For functional validation of these interactions, researchers can employ PI3K-Akt pathway inhibitors such as LY294002 , followed by assessment of changes in PIK3AP1 phosphorylation status and subcellular localization. Additionally, proximity-based assays such as fluorescence resonance energy transfer (FRET) or proximity ligation assay (PLA) can provide direct evidence of molecular interactions between PIK3AP1 and PI3K components in both fixed samples and living B cells during receptor activation processes.

What are the optimal fixation and permeabilization methods for intracellular detection of PIK3AP1?

Optimizing fixation and permeabilization protocols is essential for successful intracellular detection of PIK3AP1 using FITC-conjugated antibodies. The protocol must balance preservation of the antigen epitope with maintenance of FITC fluorescence and adequate cellular permeabilization. Based on recommended applications for immunofluorescence techniques (IF/IHC-P, IF/IHC-F, IF/ICC) , a standard approach involves fixation with 4% paraformaldehyde for 10-15 minutes at room temperature, which effectively preserves cellular architecture while maintaining epitope accessibility.

For permeabilization, a moderate concentration of 0.1-0.3% Triton X-100 applied for 5-10 minutes provides sufficient access to cytoplasmic PIK3AP1 without excessive protein extraction. Since PIK3AP1 displays both cytoplasmic and membrane localization patterns , overly harsh permeabilization conditions should be avoided as they may disrupt membrane-associated protein pools. For flow cytometry applications targeting phosphorylated forms of PIK3AP1, methanol-based fixation/permeabilization (80% methanol, -20°C, 15 minutes) may yield superior results by better exposing phospho-epitopes.

In all experimental designs, samples should be blocked with 5% BSA or appropriate normal serum prior to antibody incubation, with the FITC-conjugated PIK3AP1 antibody diluted within the manufacturer-recommended range (1:50-200 for immunofluorescence applications) .

How does PIK3AP1 expression change during B-cell development and activation?

PIK3AP1 expression undergoes dynamic regulation during B-cell development and activation processes, reflecting its critical function in B-cell signaling networks. While the search results don't provide comprehensive developmental expression profiles, they clearly highlight PIK3AP1's importance in B-cell development through its role linking B-cell receptor (BCR) signaling to the PI3K-Akt pathway . During B-cell activation, PIK3AP1 becomes tyrosine phosphorylated by kinases including Syk, Btk, or Lyn, creating binding sites for the p85 regulatory subunit of PI3K . This phosphorylation event represents a crucial step in coupling BCR signaling to downstream PI3K activation.

PIK3AP1 has also been implicated in mature B-cell survival through activation of the REL transcription factor , suggesting potential expression and activity increases during specific maturation stages. To comprehensively characterize these changes, researchers can employ the PIK3AP1 Antibody, FITC conjugated in flow cytometry analyses of B cells at different developmental stages (pro-B, pre-B, immature, and mature B cells) or following activation with various stimuli (anti-IgM, CD40L, IL-4, etc.). This methodological approach enables quantification of both expression levels and subcellular localization changes throughout B-cell developmental progression and during activation responses.

What are the best methods to validate PIK3AP1 antibody specificity?

Validating PIK3AP1 antibody specificity requires implementing multiple complementary approaches to ensure experimental reliability. Western blotting represents a fundamental validation technique, using lysates from PIK3AP1-expressing and non-expressing cells to confirm detection of bands at the expected molecular weight (approximately 90-100 kDa). The search results recommend western blot applications at dilutions of 1:300-5000 .

Genetic validation provides strong evidence of specificity and can be achieved through siRNA-mediated knockdown or CRISPR/Cas9 knockout of PIK3AP1, which should result in proportionally reduced or completely absent antibody signal. Complementary to knockdown approaches, overexpression systems utilizing appropriate expression vectors (such as the pcDNA3.1 vector system mentioned in the literature) serve as valuable positive controls for antibody validation.

Peptide blocking experiments, where pre-incubation of the antibody with the immunizing peptide should eliminate specific staining, provide additional validation. For immunofluorescence applications, co-localization with other well-characterized B-cell signaling components offers further confirmatory evidence. When possible, researchers should compare results obtained using different antibody clones targeting distinct epitopes of PIK3AP1, as this cross-validation approach significantly strengthens confidence in experimental observations.

How can researchers differentiate between membrane-bound and cytoplasmic PIK3AP1?

Distinguishing between membrane-associated and cytoplasmic pools of PIK3AP1 requires carefully optimized immunofluorescence protocols and appropriate imaging technologies. According to the search results, PIK3AP1 exhibits subcellular localization in both cytoplasmic and membrane compartments , presenting a technical challenge for precise localization studies. To address this challenge, confocal microscopy with z-stack acquisition provides superior resolution of membrane versus cytoplasmic signals compared to standard widefield fluorescence approaches.

Implementing co-staining protocols with established membrane markers (such as wheat germ agglutinin for general membrane visualization or CD19 for B-cell membranes) enables precise delineation of the plasma membrane boundary. For more selective membrane visualization, researchers can employ differential permeabilization techniques: gentle detergents like digitonin (0.001-0.005%) preferentially permeabilize the plasma membrane while leaving intracellular membranes intact, allowing selective access to membrane-associated PIK3AP1 pools.

To complement imaging approaches, subcellular fractionation followed by western blotting provides quantitative assessment of PIK3AP1 distribution between membrane and cytoplasmic compartments. When using the PIK3AP1 Antibody, FITC conjugated for these applications, researchers should carefully optimize dilutions within the recommended range (1:50-200 for immunofluorescence) and adjust imaging parameters to account for the typically lower signal intensity of membrane-bound proteins compared to concentrated cytoplasmic pools.

What role does PIK3AP1 play in viral infection models?

PIK3AP1 has emerged as an important factor in viral infection processes, with recent research revealing both direct and pathway-mediated effects on viral replication. According to published findings, "Overexpression of PIK3AP1 inhibits ASFV (African swine fever virus) replication in a way independent of the PI3K-Akt pathway" . This observation suggests that PIK3AP1 possesses antiviral properties that operate through mechanisms distinct from its canonical role in PI3K-Akt signaling.

Interestingly, viral proteins directly interact with PIK3AP1, as evidenced by the discovery that "ASFV MGF360-9L interacts with PIK3AP1 and decreases its protein expression level" . This finding indicates that certain viruses may have evolved strategies to counteract PIK3AP1's antiviral functions by modulating its expression or activity. The PI3K-Akt pathway itself has been identified "as a positive regulator on genotype II ASFV replication in natural host cells" , presenting a complex scenario where PIK3AP1 may have both pathway-dependent and pathway-independent effects during viral infection.

To investigate these mechanisms, researchers can utilize PIK3AP1 Antibody, FITC conjugated for tracking PIK3AP1 localization changes during infection, quantifying expression levels in infected versus uninfected cells, and monitoring co-localization with viral proteins through multicolor imaging approaches. These studies can help elucidate whether PIK3AP1's antiviral effects stem from direct interactions with viral components or through modulation of immune signaling pathways.

What are the technical considerations for multiplexing PIK3AP1, FITC antibody with other fluorophore-conjugated antibodies?

When designing multiplex staining panels incorporating PIK3AP1 Antibody, FITC conjugated with other fluorophore-labeled antibodies, several technical considerations must be addressed to ensure accurate data acquisition and interpretation. First, spectral compatibility requires careful planning: FITC has excitation/emission maxima at approximately 495/519 nm, so companion fluorophores should have minimal spectral overlap (appropriate choices include PE, APC, or far-red dyes like Cy5).

For flow cytometry applications, single-stained compensation controls for each fluorophore are essential to mathematically correct for spectral overlap between channels. The relative abundance of target proteins should guide panel design—PIK3AP1 might benefit from detection with FITC (a relatively bright fluorophore) if expressed at moderate levels, while more abundant proteins could be detected with dimmer fluorophores to balance signal intensities across channels.

Physical constraints of antibody binding must be considered—when studying proteins that interact with PIK3AP1 (such as PI3K subunits mentioned in the literature) , steric hindrance might affect binding efficiency. Fixation and permeabilization protocols must be compatible with all antibodies in the panel, considering the storage buffer conditions mentioned in the product specifications (containing 50% glycerol and 0.01M PBS/TBS) . If antibodies from the same host species are used in the panel, sequential staining protocols may be necessary to avoid cross-reactivity issues.

How does blocking the PI3K-Akt pathway affect PIK3AP1 phosphorylation and detection?

Inhibition of the PI3K-Akt pathway has significant implications for PIK3AP1 phosphorylation status and detection in experimental systems. The literature indicates that PIK3AP1 functions as a signaling adapter linking B-cell receptor (BCR) signaling to the PI3K-Akt pathway and becomes phosphorylated by kinases including Syk, Btk, or Lyn to provide binding sites for the p85 subunit of PI3K . When employing PI3K inhibitors such as LY294002 (mentioned in the research literature) , researchers should anticipate several effects on PIK3AP1 biology.

While the initial phosphorylation of PIK3AP1 may not be directly affected since it occurs upstream of PI3K activity, subsequent signaling events and potential feedback loops that influence PIK3AP1 phosphorylation state would likely be disrupted. The subcellular localization of PIK3AP1 might change following pathway inhibition, potentially altering detection patterns in imaging applications. Treatment with pathway inhibitors could reveal differential regulation of PIK3AP1 isoforms or post-translational modifications not evident under normal signaling conditions.

Interestingly, research findings indicate that "the suppressive effect of PIK3AP1 on ASFV replication was independent of the PI3K-Akt pathway" , suggesting complex regulatory relationships beyond the canonical pathway. For comprehensive assessment, experimental designs should include time-course analyses after inhibitor treatment and employ phospho-specific antibodies alongside total PIK3AP1 detection to monitor both expression levels and phosphorylation status changes.