PIK3CA Antibody, FITC conjugated

What is the molecular function of PIK3CA and why is it an important research target?

PIK3CA (Phosphoinositide-3-Kinase, Catalytic, alpha Polypeptide) functions as a critical enzyme that phosphorylates various phosphatidylinositol substrates including PtdIns, PtdIns4P, and PtdIns(4,5)P2 to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). This catalytic activity represents a pivotal event in cellular signaling cascades. PIP3 serves as a second messenger that recruits PH domain-containing proteins to the membrane, particularly AKT1 and PDPK1, thereby initiating downstream signaling events essential for cell growth, survival, proliferation, motility, and morphology regulation . PIK3CA participates in cellular responses to multiple growth factors and is critically involved in the activation of AKT1 following stimulation by various receptor tyrosine kinase ligands including EGF, insulin, IGF1, VEGFA, and PDGF . The protein is widely expressed across numerous tissue types, making it a valuable target for studying signaling pathway dysregulation in various physiological and pathological contexts .

What specific applications are validated for PIK3CA Antibody, FITC conjugated?

PIK3CA Antibody, FITC conjugated has been validated for multiple experimental applications that leverage both its target specificity and fluorescent properties:

Importantly, Western Blot represents one of the most widely used applications for PIK3CA antibodies, with over 990 citations in scientific literature documenting successful implementation . Immunohistochemistry also remains a common application for these antibodies in research settings .

What is the species reactivity profile of commercially available PIK3CA Antibody, FITC conjugated?

The reactivity profile of PIK3CA Antibody, FITC conjugated varies slightly between manufacturers but generally exhibits broad cross-reactivity across multiple species:

The PIK3CA gene exhibits significant conservation across species, with orthologs reported in numerous vertebrates including mammals, birds, and fish . This conservation facilitates comparative studies of PI3K signaling across evolutionary diverse organisms.

What are the optimal storage conditions for maintaining PIK3CA Antibody, FITC conjugated activity?

Proper storage of PIK3CA Antibody, FITC conjugated is essential for maintaining its specificity, sensitivity, and fluorescent signal integrity. According to manufacturer specifications, the following storage protocol is recommended:

Storage temperature should be maintained at -20°C to prevent protein degradation and preserve fluorophore activity . Multiple freeze-thaw cycles significantly reduce antibody performance; therefore, researchers should aliquot the stock solution into multiple small-volume vials to avoid repeated thawing of the entire stock . The antibody formulation typically includes stabilizing components such as 50% glycerol, 0.01M TBS (pH 7.4), 1% BSA, and 0.03% Proclin300, which collectively help maintain antibody structure and activity during storage .

For FITC-conjugated antibodies specifically, protection from light exposure during both storage and handling is critical to prevent photobleaching of the fluorophore. Extended exposure to ambient light can significantly diminish signal intensity in subsequent applications.

How can PIK3CA Antibody, FITC conjugated be optimized for phosphorylation-specific signaling studies?

When investigating PI3K pathway activation through phosphorylation events, researchers must carefully consider several optimization parameters:

The PI3K signaling cascade involves multiple phosphorylation steps, including the phosphorylation of regulatory subunits such as p85α at Y467 which modulates catalytic activity . For comprehensive pathway analysis, researchers should consider using both phospho-specific and total protein antibodies. For instance, antibodies targeting phosphorylated p85α (Y467) can be used alongside PIK3CA antibodies to assess activation states relative to total protein levels .

For cell stimulation experiments, time-course studies ranging from acute (5-15 minutes) to prolonged (24-48 hours) exposure to growth factors such as EGF, insulin, or PDGF should be conducted to capture the temporal dynamics of phosphorylation events. Pharmacological inhibitors like wortmannin or LY294002 serve as essential controls to confirm signal specificity through the PI3K pathway.

When using fluorescent conjugates like FITC for phospho-signaling studies, researchers should normalize signal intensities to account for variations in total protein expression between experimental conditions. This is particularly important when investigating mutations that may affect both phosphorylation status and protein stability.

What methodological approaches enhance PIK3CA detection in flow cytometry applications?

Flow cytometry using PIK3CA Antibody, FITC conjugated requires specific technical considerations to achieve optimal results:

For intracellular staining of PIK3CA, robust fixation and permeabilization protocols are essential due to its predominantly cytoplasmic localization . A two-step procedure using 4% paraformaldehyde for initial fixation followed by permeabilization with either 0.1% Triton X-100 or commercially available permeabilization buffers typically yields reliable results.

Titration experiments should be performed to determine optimal antibody concentration, typically starting with the manufacturer's recommended dilution (often 1:50 to 1:200) and adjusting based on signal-to-noise ratio. For FITC-conjugated antibodies, compensation controls are necessary when designing multicolor panels to account for spectral overlap with other fluorophores.

Cell cycle synchronization may improve detection consistency, as PIK3CA expression and activation can fluctuate throughout the cell cycle. Serum starvation followed by stimulation with growth factors provides a controlled system for analyzing pathway dynamics. Representative results from optimal and suboptimal flow cytometry procedures should include histogram overlays demonstrating shifts in fluorescence intensity following pathway stimulation.

How do different fixation protocols affect immunofluorescence results with PIK3CA Antibody, FITC conjugated?

Fixation methodology significantly impacts immunofluorescence outcomes with PIK3CA Antibody, FITC conjugated:

For paraffin-embedded tissues (IHC-P), heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is often necessary to unmask epitopes following formalin fixation . For frozen tissue sections (IHC-F), brief fixation in cold acetone typically provides adequate results while preserving tissue morphology . Counterstaining with DAPI allows nuclear visualization, which is helpful in determining subcellular localization of PIK3CA, which is primarily cytoplasmic .

What validation approaches confirm specificity of PIK3CA Antibody, FITC conjugated in experimental systems?

Comprehensive validation of PIK3CA Antibody, FITC conjugated should include:

Genetic approaches using PIK3CA knockout or knockdown models provide the gold standard for antibody validation. Comparing staining patterns between wild-type cells and those with CRISPR/Cas9-mediated PIK3CA deletion or siRNA-mediated knockdown confirms specificity. Peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific staining, offer another validation method.

Molecular weight verification is essential, with human PIK3CA expected at approximately 124.3 kDa . Multiple detection methods should yield consistent results across Western blot, immunofluorescence, and flow cytometry applications. Correlation with mRNA expression data from qPCR or RNA-seq provides additional validation of antibody specificity across different experimental contexts.

For phospho-specific antibodies in the PI3K pathway, treatment with phosphatase inhibitors should enhance signal, while phosphatase treatment should diminish it, confirming phospho-specificity of the detected epitope .

How can PIK3CA Antibody, FITC conjugated contribute to cancer research applications?

PIK3CA Antibody, FITC conjugated offers significant value in cancer research contexts:

The PIK3CA gene is frequently mutated in multiple cancer types, particularly colorectal cancer, with mutations concentrated in hotspot regions that enhance kinase activity and drive oncogenic signaling . FITC-conjugated antibodies enable direct visualization of PIK3CA protein expression and localization in tumor samples, facilitating correlation with mutation status and clinical outcomes.

In cell line models harboring PIK3CA mutations, flow cytometry with PIK3CA Antibody, FITC conjugated can measure changes in protein expression following treatment with PI3K pathway inhibitors, providing pharmacodynamic readouts for drug efficacy. Multiplexed immunofluorescence combining PIK3CA detection with markers of downstream pathway activation (phospho-AKT, phospho-S6) enables comprehensive pathway analysis in heterogeneous tumor tissues.

Co-localization studies with receptor tyrosine kinases allow investigation of membrane recruitment dynamics, while xenograft models can be analyzed using immunofluorescence to assess drug effects on PIK3CA expression and pathway activity in vivo. The cytoskeletal organization and adipose tissue development functions of PIK3CA make it particularly relevant in studying cancer cell invasion and metabolic adaptations during tumorigenesis .

What optimization parameters improve Western blot detection using PIK3CA Antibody, FITC conjugated?

Western blot optimization for PIK3CA Antibody, FITC conjugated should address:

Sample preparation is critical, with complete cell lysis using RIPA or NP-40 buffers containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride) and protease inhibitors necessary to preserve PIK3CA integrity. Protein loading should be standardized at 20-50 μg per lane, with gradient gels (4-12%) often providing superior resolution for the 124.3 kDa PIK3CA protein .

While FITC conjugation enables direct fluorescent detection, researchers should note that chemiluminescent detection using HRP-conjugated secondary antibodies generally provides greater sensitivity for Western blot. For fluorescent detection, low-autofluorescence PVDF membranes are preferable to nitrocellulose.

Blocking should utilize 5% BSA rather than milk, as milk contains phosphoproteins that may interfere with phospho-epitope detection in pathway analysis. Transfer conditions should be optimized for high molecular weight proteins (124.3 kDa), typically using lower current for extended periods to ensure complete transfer.

What strategies address non-specific binding in immunohistochemistry with PIK3CA Antibody, FITC conjugated?

To minimize non-specific binding in immunohistochemistry:

Thorough blocking with species-appropriate serum (5-10%) combined with BSA (1-3%) significantly reduces background. For tissue samples with high endogenous biotin, avidin-biotin blocking steps should be incorporated before antibody application even when using directly conjugated FITC antibodies.

Antibody titration is essential, with typical working dilutions ranging from 1:50 to 1:200, though optimal concentration must be empirically determined for each tissue type. Washing steps should be extended (3x15 minutes) and performed with gentle agitation to effectively remove unbound antibody.

Autofluorescence can be particularly problematic in FITC-based detection. Treatment with Sudan Black B (0.1-0.3% in 70% ethanol) or commercial autofluorescence reducers helps minimize tissue autofluorescence, particularly in tissues rich in lipofuscin or collagen. Including an isotype control antibody conjugated to FITC helps distinguish specific staining from non-specific binding or autofluorescence.

How should researchers approach multiplex immunofluorescence assays incorporating PIK3CA Antibody, FITC conjugated?

Effective multiplex immunofluorescence strategies should consider:

Antibody panel design must account for the spectral properties of FITC (excitation ~495 nm, emission ~520 nm), selecting additional fluorophores with minimal spectral overlap such as TRITC, Cy5, or APC. Sequential staining protocols often yield superior results compared to cocktail approaches, particularly when combining antibodies from the same host species.

Signal amplification techniques such as tyramide signal amplification may be necessary when target expression is low, though this requires HRP-conjugated rather than direct FITC-conjugated antibodies. Multispectral imaging platforms with spectral unmixing capabilities help resolve overlapping fluorophore emissions in complex multiplex panels.

For co-localization studies examining interactions between PIK3CA and binding partners or downstream targets, confocal microscopy with appropriate controls for bleed-through is essential. Quantitative analysis should include colocalization coefficients (Pearson's or Mander's) calculated from multiple regions of interest across independent samples.

What are the critical controls for experiments using PIK3CA Antibody, FITC conjugated?

Rigorous experimental design requires specific controls:

Positive controls should include cell lines or tissues with confirmed PIK3CA expression, such as MCF-7 breast cancer cells or HCT116 colorectal cancer cells. Negative controls should incorporate tissue or cell types with minimal PIK3CA expression, or isogenic cell lines with CRISPR/Cas9-mediated PIK3CA knockout.

Technical negative controls must include isotype controls (rabbit IgG-FITC) processed identically to experimental samples to assess non-specific binding . For stimulation experiments, both unstimulated and stimulated samples should be processed in parallel, with pathway inhibitor controls (wortmannin, LY294002) confirming signal specificity.

Antibody specificity controls should include pre-adsorption with immunizing peptide where available, which should eliminate specific staining. For FITC-conjugated antibodies specifically, photobleaching controls help distinguish true signal from autofluorescence, particularly in tissues with high intrinsic fluorescence like liver or kidney.

How do PIK3CA antibody selection criteria differ between research applications?

Selection criteria for PIK3CA antibodies should be tailored to specific research objectives:

The immunogen range is a critical consideration, with antibodies generated against different regions of PIK3CA potentially yielding different results. For example, antibodies targeting amino acids 1-206 may recognize different conformational states compared to those targeting the C-terminal region (amino acids 961-1068/1068) . For studies focusing on specific PIK3CA isoforms or splice variants, epitope mapping is essential to ensure detection of the relevant protein species.