PIK3AP1 Antibody, Biotin conjugated

What is PIK3AP1 and what cellular functions does it perform?

PIK3AP1 (phosphoinositide-3-kinase adaptor protein 1), also known as BCAP (B-cell adaptor for PI3K), is a specific protein adaptor primarily expressed in hematopoietic cells. It plays crucial roles in:

• Activation of phosphoinositide 3-kinase (PI3K) in B-cells and natural killer (NK) cells

• Coupling B-cell antigen receptor (BCR) to PI3K activation by providing a docking site for the PI3K subunit PIK3R1

• Contributing to B-cell development through complementary interactions with CD19 in PI3K activation

• Supporting survival of mature B cells via activation of REL (by similarity)

• Coordinating antigen responses in immune cells

The protein has a predicted molecular weight of 90.2 kDa and functions within the B cell receptor signaling pathway, making it an important target for immunological research .

What applications are supported by biotin-conjugated PIK3AP1 antibodies?

Biotin-conjugated PIK3AP1 antibodies support multiple research applications with specific optimization parameters:

• Western Blot (WB): Recommended dilution of 1:2000, allowing detection of the ~90 kDa PIK3AP1 protein

• Immunofluorescence (IF): Recommended dilution of 1:100 for optimal signal-to-noise ratio

• Flow Cytometry (FC): Recommended dilution of 1:100 for cell surface and intracellular detection

The biotin conjugation provides significant advantages for detection sensitivity through streptavidin-based amplification systems, particularly in complex experimental setups involving multiple antibodies .

What is the proper handling and storage protocol for maintaining antibody activity?

To maintain optimal activity of the biotin-conjugated PIK3AP1 antibody:

• Store at -20°C as received in the original container

• The antibody formulation (PBS at pH 7.3 containing 1% BSA, 50% glycerol, and 0.02% sodium azide) is designed for long-term stability

• For antibodies at 0.5 mg/ml concentration, aliquoting is generally unnecessary for -20°C storage

• Avoid repeated freeze-thaw cycles to prevent degradation of the antibody and loss of biotin activity

• Maintain appropriate documentation of thawing dates and storage conditions

Proper storage ensures continued reactivity with human, mouse, and rat samples over the expected shelf life of the product .

How should Western blot protocols be optimized for PIK3AP1 detection?

For optimal Western blot results with PIK3AP1 antibody:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Load 20-40 μg of total protein per lane

  • Include positive control samples (e.g., HT-29 cells which have confirmed PIK3AP1 expression)

• Gel Electrophoresis and Transfer:

  • Use 8-10% SDS-PAGE gels to properly resolve the 90.2 kDa PIK3AP1 protein

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Antibody Incubation:

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

  • Dilute primary antibody at 1:2000-1:5000 (depending on expression level)

  • Incubate overnight at 4°C with gentle agitation

  • For biotin-conjugated antibodies, use streptavidin-HRP (1:10000) as secondary detection reagent

• Detection and Analysis:

  • Develop using standard ECL reagents

  • Expected band size is approximately 90 kDa

Validation studies show clear bands at the predicted molecular weight across human, mouse, and rat samples when following this protocol .

What are the critical steps for immunofluorescence experiments using PIK3AP1 antibody?

For successful immunofluorescence detection of PIK3AP1:

• Cell Preparation:

  • Culture cells on coverslips or use cytospin for suspension cells

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

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Antibody Staining:

  • Block with 1% BSA in PBS for 30 minutes

  • Dilute PIK3AP1 primary antibody 1:100 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • For biotin-conjugated antibody, use streptavidin-fluorophore conjugates for detection

  • Include DAPI (1:1000) for nuclear counterstaining

• Visualization:

  • Mount slides with anti-fade mounting medium

  • Observe predominantly cytoplasmic staining pattern with potential membrane association

  • Document exposure settings for consistent imaging

The biotin conjugation provides excellent signal amplification when paired with fluorescently labeled streptavidin, enabling detection of native protein expression levels .

How can flow cytometry protocols be optimized for PIK3AP1 detection?

For flow cytometry applications with PIK3AP1 antibody:

• Cell Preparation:

  • Harvest 1×10^6 cells per sample

  • Fix with 2% paraformaldehyde for 10 minutes at room temperature

  • Permeabilize with 0.1% saponin (for intracellular staining)

• Staining Protocol:

  • Block with 5% normal serum in staining buffer

  • Dilute biotin-conjugated PIK3AP1 antibody 1:100

  • Incubate for 30 minutes at 4°C

  • Wash twice with staining buffer

  • Incubate with streptavidin-fluorophore (e.g., streptavidin-PE or streptavidin-APC) at 1:200

  • Wash twice before analysis

• Controls and Analysis:

  • Include isotype control antibody at the same concentration

  • Use single-stained controls for compensation

  • Gate on viable cells using appropriate viability dye

  • Analyze with standard B-cell markers (CD19, CD20) for co-expression studies

This protocol has demonstrated successful detection of PIK3AP1 in primary B cells and B cell lines with high specificity and low background .

How can PIK3AP1 antibody be used to study B-cell receptor signaling pathways?

For investigating BCR signaling using PIK3AP1 antibody:

• Stimulation Experiments:

  • Stimulate B cells with anti-IgM (10 μg/ml) for various time points (0, 5, 15, 30 min)

  • Lyse cells and perform immunoprecipitation with anti-PIK3AP1

  • Analyze co-precipitating proteins by Western blot using phospho-specific antibodies

• Proximity Ligation Assay:

  • Fix stimulated cells on slides

  • Use PIK3AP1 antibody alongside antibodies against BCR signaling components (e.g., CD19, PIK3R1)

  • Follow manufacturer's protocol for proximity ligation

  • Quantify interaction spots by confocal microscopy

• Functional Assays:

  • Compare PI3K activation (measured by pAKT) in control versus PIK3AP1-depleted cells

  • Analyze calcium mobilization following BCR stimulation

  • Assess F-actin reorganization using phalloidin staining

Research has shown that PIK3AP1 coordinates antigen responses by functioning as a critical adapter that couples BCR engagement with downstream PI3K activation. The biotin-conjugated antibody is particularly useful for multicolor flow cytometry experiments examining signaling dynamics .

What methodologies are recommended for studying PIK3AP1 in cancer research models?

For cancer research applications focusing on PIK3AP1:

• Expression Analysis:

  • Compare PIK3AP1 levels across cancer cell lines using Western blot

  • Standardize loading with β-actin (recommended dilution 1:5000)

  • Quantify expression relative to normal tissue controls

• miRNA Regulation Studies:

  • Examine the miR-567-PIK3AP1-PI3K/AKT-c-Myc feedback loop in gastric cancer

  • Transfect cells with miR-567 mimics/inhibitors

  • Measure PIK3AP1 expression changes by Western blot

  • Assess downstream PI3K/AKT activation

• Functional Assays:

  • Perform knockdown/overexpression of PIK3AP1 in cancer cell lines

  • Evaluate effects on proliferation, migration, and chemoresistance

  • Analyze PI3K pathway activation using phospho-specific antibodies

  • Combine with patient sample analysis for clinical relevance

Studies have implicated PIK3AP1 in gastric cancer development through its role in the PI3K/AKT signaling pathway, making it a potentially important biomarker and therapeutic target .

How can researchers assess interactions between PIK3AP1 and cytoskeletal proteins?

To investigate PIK3AP1 interactions with cytoskeletal components:

• Co-immunoprecipitation:

  • Lyse B cells or macrophages in non-denaturing buffer

  • Immunoprecipitate with PIK3AP1 antibody

  • Probe Western blots for actin-regulatory proteins (Flightless-1, L-plastin, gelsolin, Arp2/3 complex)

• Immunofluorescence Co-localization:

  • Fix cells following BCR stimulation

  • Co-stain with PIK3AP1 antibody and phalloidin for F-actin

  • Use confocal microscopy to assess co-localization at different time points

  • Quantify F-actin clearance around the antigen

• Live Cell Imaging:

  • Transfect cells with fluorescently-tagged PIK3AP1

  • Perform live imaging during B cell activation

  • Measure recruitment kinetics of PIK3AP1 to the immunological synapse

  • Correlate with actin dynamics

Research has demonstrated that PIK3AP1 interacts with several F-actin-regulatory proteins in macrophages and may serve as a polarity cue to attract the centrosome during BCR engagement, facilitating F-actin clearance around the antigen .

What might cause multiple bands in Western blot using PIK3AP1 antibody?

When troubleshooting multiple bands in PIK3AP1 Western blots:

• Expected Patterns and Explanations:

  • Primary band at ~90 kDa (full-length protein)

  • Lower molecular weight bands may represent:

    • Proteolytic degradation products (improve sample preparation)

    • Alternative splice variants (verify in literature)

    • Post-translational modifications (consider phosphatase treatment)

• Technical Solutions:

  • Add additional protease inhibitors to lysis buffer

  • Reduce sample processing time and maintain cold temperatures

  • Optimize gel percentage (8-10% recommended)

  • Adjust antibody concentration (try 1:5000-1:50000 dilution range)

  • Include positive control samples (HT-29 cells recommended)

• Validation Approaches:

  • Use siRNA knockdown to confirm specificity of bands

  • Compare with alternative antibody clones

  • Preincubate antibody with immunizing peptide

Quality control data shows that when properly optimized, the PIK3AP1 antibody should produce a predominant band at the expected 90 kDa molecular weight in most cell types, with minimal non-specific binding .

How should researchers address cross-reactivity concerns with PIK3AP1 antibody?

To address potential cross-reactivity issues:

• Species Cross-Reactivity Assessment:

  • The antibody shows confirmed reactivity with human, mouse, and rat samples

  • When using in other species, perform validation experiments:

    • Western blot comparison with positive control samples

    • Sequencing alignment of the immunogen region across species

• Isotype Controls:

  • Use isotype-matched control antibodies at the same concentration

  • For mouse monoclonal PIK3AP1 antibody (OTI5H5), use mouse IgG

  • Include in all applications (WB, IF, FC) to identify non-specific binding

• Antibody Validation Techniques:

  • Genetic approaches: Test in PIK3AP1 knockout/knockdown systems

  • Biochemical approaches: Preabsorption with recombinant PIK3AP1

  • Orthogonal approaches: Compare with alternative detection methods

The specificity of clone OTI5H5 has been validated against full-length human recombinant PIK3AP1 protein (NP_689552) produced in HEK293T cells, providing confidence in target recognition .

What are the critical quality control parameters for antibody validation?

Essential quality control parameters for PIK3AP1 antibody validation include:

For the biotin-conjugated variant specifically:

• Confirm biotin activity using streptavidin detection systems

• Assess potential stericity issues that might affect epitope binding

• Verify conjugation ratio is optimal for detection without compromising antibody activity

Professional quality control protocols include purification by affinity chromatography (protein A/G) to ensure high specificity and consistency between production batches .

How can PIK3AP1 antibody be utilized in studying immune cell polarization and migration?

For investigating immune cell polarization and migration:

• Live Cell Imaging Protocol:

  • Label cells with biotin-conjugated PIK3AP1 antibody followed by fluorescent streptavidin

  • Perform time-lapse imaging during chemotactic migration

  • Quantify PIK3AP1 localization during cell polarization

• Actin Cytoskeleton Co-visualization:

  • Co-stain PIK3AP1 with F-actin markers

  • Analyze redistribution during immune synapse formation

  • Quantify F-actin clearance dynamics

  • Compare control and PIK3AP1-deficient cells

• Centrosome Recruitment Analysis:

  • Use PIK3AP1 antibody alongside centrosome markers

  • Measure kinetics of centrosome polarization

  • Assess recruitment of actin regulators to the antigen area

Recent research indicates that PIK3AP1 acts as a polarity cue to attract the centrosome during BCR engagement, which then concentrates actin regulators to facilitate F-actin clearance around the antigen. This plays a crucial role in immune cell function and response to pathogens .

What are the current methods for studying PIK3AP1 in the context of PI3K pathway activation?

To investigate PIK3AP1's role in PI3K pathway activation:

• Phosphorylation Cascade Analysis:

  • Stimulate cells (e.g., with anti-IgM for B cells)

  • Immunoprecipitate PIK3AP1 using the antibody

  • Probe for phosphorylated residues with anti-pTyr antibodies

  • Assess recruitment of PI3K subunits (PIK3R1)

• Downstream Signaling Measurement:

  • Monitor phosphorylation of AKT (Ser473, Thr308)

  • Assess activation of mTOR pathway components

  • Quantify PIP3 production using specific biosensors

  • Compare signaling kinetics in control versus PIK3AP1-deficient cells

• Genetic Complementation Studies:

  • Use PIK3AP1 knockout/conditional knockout models

  • Reconstitute with wild-type or mutant PIK3AP1

  • Analyze restoration of PI3K pathway activation

  • Measure functional outcomes in B cell development and activation

PIK3AP1 has been shown to play a critical role in coupling B-cell antigen receptor (BCR) to PI3K activation by providing a docking site for the PI3K subunit PIK3R1, which contributes significantly to B-cell development. Recent studies suggest a complementary role with CD19 in this process .

How can PIK3AP1 antibody be used in ELISA-based quantification methods?

For ELISA-based quantification of PIK3AP1:

• Sandwich ELISA Protocol:

  • Coat microwells with capture antibody against PIK3AP1

  • Block with appropriate buffer

  • Add samples and standards

  • Detect with biotin-conjugated PIK3AP1 antibody (OTI5H5)

  • Develop with streptavidin-HRP and TMB substrate

  • Measure absorbance at 450 nm

• Sample Preparation Guidance:

  • For serum/plasma: Dilute samples appropriately based on expected concentration

  • For cell lysates: Use non-denaturing lysis buffers

  • Perform serial dilutions to ensure readings fall within standard curve

  • Include quality control samples of known concentration

• Data Analysis Considerations:

  • Generate standard curve using recombinant PIK3AP1

  • Perform regression analysis (4-parameter logistic curve recommended)

  • Calculate concentration of unknown samples

  • Account for dilution factors in final calculations

Commercial ELISA systems utilizing sandwich technique with monoclonal antibodies demonstrate higher specificity and increased sensitivity compared to conventional competitive ELISA kits that employ only one antibody. The biotin-conjugated antibody provides excellent sensitivity in this application .

How does PIK3AP1 contribute to B cell-mediated pathologies?

PIK3AP1's role in B cell-mediated diseases includes:

• Autoimmune Disease Mechanisms:

  • PIK3AP1 regulates B cell survival through PI3K pathway activation

  • Dysregulation may contribute to:

    • Hyperactive B cell responses

    • Enhanced autoantibody production

    • Altered B cell selection in germinal centers

• Experimental Assessment Approaches:

  • Compare PIK3AP1 expression/activation in patient samples versus controls

  • Analyze signaling in B cells from autoimmune disease models

  • Use conditional knockout models to assess disease development

  • Test therapeutic targeting of PIK3AP1-dependent pathways

• Potential Therapeutic Implications:

  • PI3K pathway inhibitors may target PIK3AP1-mediated signaling

  • Selective modulation of PIK3AP1 could provide B cell-specific intervention

  • Biomarker potential for stratifying patients for PI3K-targeted therapies

Research suggests PIK3AP1 may be involved in the survival of mature B cells via activation of REL, pointing to potential roles in B cell-mediated pathologies where excessive B cell survival contributes to disease progression .

What is known about the role of PIK3AP1 in gastric cancer development?

Current understanding of PIK3AP1 in gastric cancer includes:

• Molecular Mechanisms:

  • PIK3AP1 functions within the miR-567-PIK3AP1-PI3K/AKT-c-Myc feedback loop

  • This regulatory network influences:

    • Tumor cell proliferation

    • Cancer progression

    • Chemoresistance development

• Experimental Detection Methods:

  • Immunohistochemistry of patient samples using PIK3AP1 antibody

  • Western blot analysis of gastric cancer cell lines

  • qRT-PCR for mRNA expression analysis

  • Functional studies using knockdown/overexpression approaches

• Clinical Correlations:

  • Expression levels may correlate with:

    • Disease progression

    • Treatment response

    • Patient prognosis

  • Potential use as biomarker for PI3K pathway activation in tumors

Studies have implicated PIK3AP1 in gastric cancer development through its role in regulating PI3K/AKT signaling, positioning it as a potential therapeutic target and prognostic indicator in gastric malignancies .

What experimental approaches can assess PIK3AP1 inhibition as a therapeutic strategy?

For evaluating PIK3AP1 as a therapeutic target:

• Genetic Inhibition Strategies:

  • siRNA/shRNA knockdown in relevant cell lines

  • CRISPR/Cas9 gene editing to create knockout models

  • Conditional knockout mouse models for in vivo studies

• Assays for Measuring Therapeutic Effect:

  • Cell proliferation (MTT/XTT assays)

  • Apoptosis assessment (Annexin V/PI staining)

  • Migration/invasion capability (Transwell assays)

  • In vivo tumor growth in xenograft models

• Combination Therapy Assessment:

  • PIK3AP1 inhibition + conventional chemotherapeutics

  • PIK3AP1 inhibition + other targeted therapies

  • PIK3AP1 inhibition + immunotherapy

  • Monitoring of synergistic effects using combination index analysis

• Biomarker Identification:

  • PIK3AP1 expression analysis in responder vs. non-responder populations

  • Correlation with PI3K pathway activation markers

  • Development of companion diagnostics using validated antibodies

Research suggests targeting the miR-567-PIK3AP1-PI3K/AKT-c-Myc feedback loop might represent a novel therapeutic strategy for gastric cancer, potentially addressing issues of chemoresistance in this challenging malignancy .