PIK3R3 Antibody

What is PIK3R3 and what cellular functions does it regulate?

PIK3R3, also known as p55PIK, is a 55 kDa regulatory subunit of phosphatidylinositol 3-kinase. It functions primarily by binding to activated (phosphorylated) protein-tyrosine kinases through its SH2 domain, thereby regulating their kinase activity. During insulin stimulation, PIK3R3 interacts with IRS-1 (Insulin Receptor Substrate 1), facilitating downstream signaling cascades . As a component of the PI3K heterodimer, PIK3R3 works with the p110 catalytic subunit to control various cellular processes including cell growth, proliferation, and survival .

PIK3R3 expression varies across tissues, with highest levels detected in brain and testis, and moderate expression in adipose tissue, kidney, heart, lung, and skeletal muscle . Recent studies have demonstrated that PIK3R3 is significantly upregulated in several cancers, including liver and ovarian cancers, where it promotes tumor growth by activating Akt signaling pathways .

What types of PIK3R3 antibodies are available and how do they differ?

Several types of PIK3R3 antibodies are available for research applications, varying in their target epitopes, host species, and validated applications:

The selection of an appropriate antibody depends on your experimental requirements, including the species being studied, application method, and the specific region of the protein you aim to detect . Antibodies targeting different epitopes may yield varying results, particularly if post-translational modifications or protein interactions affect epitope accessibility.

How should PIK3R3 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of PIK3R3 antibodies are essential for maintaining their specificity and sensitivity:

For short-term storage (up to 2 weeks), antibodies should be maintained at 2-8°C . For long-term storage, keep antibodies at -20°C in small aliquots to prevent freeze-thaw cycles that can degrade antibody quality . Most PIK3R3 antibodies are supplied in PBS with 0.09% (W/V) sodium azide as a preservative and may be prepared by Saturated Ammonium Sulfate (SAS) precipitation followed by dialysis against PBS or through affinity purification methods .

When handling PIK3R3 antibodies, follow these practices:

• Briefly centrifuge vials before opening to collect liquid

• Minimize exposure to light when working with fluorophore-conjugated antibodies

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

• Always wear appropriate personal protective equipment due to the presence of sodium azide, which is toxic and hazardous

• Remember that PIK3R3 antibodies are for research use only and not for diagnostic or therapeutic applications

What are the optimal dilutions for using PIK3R3 antibodies in various applications?

The optimal dilution of PIK3R3 antibodies varies by application and specific antibody formulation:

These dilutions should be considered starting points rather than definitive values . Optimal concentrations should be determined experimentally for each specific antibody, sample type, and detection system. Factors affecting optimal dilution include PIK3R3 abundance in your sample, sensitivity of your detection system, and quality of sample preparation.

What are the best validation strategies for confirming PIK3R3 antibody specificity?

Validating antibody specificity is crucial for ensuring reliable experimental results. For PIK3R3 antibodies, consider these validation approaches:

• Positive and negative control samples:

  • Use tissues known to express high levels of PIK3R3 (brain, testis) as positive controls

  • Include tissues with minimal expression or PIK3R3 knockdown cells as negative controls

  • Compare results across multiple sample types to confirm expected expression patterns

• Multiple detection methods:

  • Confirm a single band at the expected molecular weight (~54.4 kDa) in Western blot

  • Validate with qRT-PCR to correlate protein expression with mRNA levels

  • Use immunohistochemistry to confirm appropriate tissue localization patterns

• Genetic manipulation:

  • Perform siRNA knockdown or CRISPR-based deletion of PIK3R3

  • Observe corresponding reduction in antibody signal intensity

  • Complement with PIK3R3 overexpression studies to confirm increased signal detection

• Advanced validation techniques:

  • Conduct peptide competition assays by pre-incubating the antibody with immunizing peptide

  • Perform immunoprecipitation followed by mass spectrometry to confirm protein identity

  • Compare results using multiple antibodies targeting different PIK3R3 epitopes

Research on liver cancer has utilized PIK3R3 knockdown validation, demonstrating significant reduction in protein expression following siRNA treatment, confirming antibody specificity while simultaneously investigating the functional impact of reduced PIK3R3 expression .

How can I optimize immunohistochemical detection of PIK3R3 in tissue samples?

Optimizing immunohistochemical detection of PIK3R3 requires careful consideration of several parameters:

• Tissue preparation and fixation:

  • Use 10% neutral buffered formalin for 24-48 hours for consistent fixation

  • Maintain consistent section thickness (4-6 μm) across experimental samples

  • Ensure complete paraffin removal and proper rehydration of sections before staining

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) provides a good starting point

  • Alternative buffers (EDTA pH 8.0 or Tris-EDTA pH 9.0) may improve detection for certain epitopes

  • Optimize retrieval duration (typically 10-30 minutes) based on fixation conditions

  • Consider using pressure cooker-based retrieval for consistent results

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with hydrogen peroxide

  • Use serum-based blocking to reduce non-specific binding

  • Optimize primary antibody dilution (starting with 1:10-1:50 for IHC-P)

  • Incubate primary antibody overnight at 4°C for improved signal-to-noise ratio

  • Include appropriate negative controls (isotype control, no primary antibody)

• Detection and signal development:

  • Select detection systems based on desired sensitivity (polymer-based systems often provide better results)

  • Optimize DAB development time to achieve optimal signal without background

  • Consider counterstaining protocols that maintain antigen visibility

• Validation and controls:

  • Include positive control tissues (brain or testis for PIK3R3)

  • Incorporate technical controls to assess non-specific binding

  • Validate findings with additional techniques (Western blotting, qRT-PCR)

Studies investigating PIK3R3 in cancer tissues have successfully employed immunohistochemistry to analyze protein expression patterns and correlate them with clinical parameters .

How can I use PIK3R3 antibodies to investigate its role in cancer progression?

PIK3R3 has emerged as a significant player in cancer biology, particularly in liver and ovarian cancers. Researchers can leverage PIK3R3 antibodies to explore its role in cancer progression through various approaches:

• Expression analysis in cancer tissues:

  • Compare PIK3R3 expression between tumor and adjacent normal tissues using IHC and Western blotting

  • Correlate expression levels with clinical parameters including tumor stage, grade, and patient survival

  • Develop tissue microarrays to analyze PIK3R3 expression across large patient cohorts

  • Examine PIK3R3 expression in cancer stem cell populations, as studies have shown elevated expression in spheroid cultures

• Functional pathway analysis:

  • Use Western blotting with PIK3R3 antibodies alongside phospho-specific antibodies for Akt pathway components

  • Investigate how PIK3R3 knockdown or overexpression affects downstream signaling molecules

  • Correlate PIK3R3 expression with cell cycle regulators like CDKN1C, which has been identified as significantly upregulated following PIK3R3 knockdown

  • Examine the relationship between PIK3R3 and structural maintenance of chromosomes protein SMC1A, which has been implicated in PIK3R3-regulated functions

• In vivo tumor models:

  • Generate stable PIK3R3 overexpression or knockdown cell lines for xenograft studies

  • Monitor tumor growth, volume, and proliferation markers (e.g., Ki67) in response to PIK3R3 modulation

  • Use immunohistochemistry with PIK3R3 antibodies to confirm expression in tumor tissues

Research has demonstrated that PIK3R3 overexpression significantly promotes liver cancer growth in vivo, with larger tumor volumes and increased Ki67 staining compared to controls . Similarly, elevated PIK3R3 expression has been observed in ovarian cancer, suggesting a broader role in multiple cancer types .

How can I use PIK3R3 antibodies to study protein-protein interactions in signaling pathways?

Understanding PIK3R3's protein interactions is crucial for elucidating its role in signaling networks. Several techniques utilizing PIK3R3 antibodies can help investigate these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use PIK3R3 antibodies to pull down native protein complexes

  • Analyze immunoprecipitates by Western blotting to detect potential binding partners

  • This approach can identify interactions with known partners like IRS-1 during insulin stimulation

  • Alternatively, perform reverse Co-IP using antibodies against suspected interaction partners

• Proximity-based techniques:

  • Employ proximity ligation assay (PLA) to visualize protein-protein interactions in situ

  • This technique amplifies signals only when proteins are within 30-40 nm of each other

  • Requires antibodies against both PIK3R3 and its potential interaction partners

  • Provides spatial information about interaction contexts within cells

• Immunofluorescence co-localization:

  • Perform dual immunofluorescence with PIK3R3 antibodies and antibodies against potential interaction partners

  • Analyze co-localization using confocal microscopy and appropriate statistical methods

  • Useful for generating hypotheses about potential interactions for further validation

• Interaction mapping:

  • Use a panel of antibodies targeting different PIK3R3 domains to determine interaction regions

  • Combine with deletion mutants to identify critical binding domains

  • Apply to understand how PIK3R3 participates in multi-protein complexes

Research has demonstrated that immunoprecipitation techniques can reveal indirect interactions between PIK3R3 and proteins like CDKN1C and SMC1A, suggesting complex regulatory networks in cancer cells . These interactions appear to be mediated through the Akt signaling pathway, highlighting the interconnected nature of PIK3R3-regulated processes.

What considerations are important when studying PIK3R3 in relation to PI3K/Akt signaling?

The PI3K/Akt pathway is a central regulator of cell growth, proliferation, and survival, with PIK3R3 playing a key regulatory role. When investigating PIK3R3 in this context, consider these methodological approaches:

• Integrated pathway analysis:

  • Use PIK3R3 antibodies alongside phospho-specific antibodies for key pathway components (p-Akt, p-mTOR, p-S6K)

  • Design experiments to assess both total protein levels and activation states

  • Consider the temporal dynamics of pathway activation following stimulus exposure

  • Examine how PIK3R3 knockdown or overexpression affects downstream signaling events

• Stimulus-response experiments:

  • Monitor PIK3R3 expression and localization changes in response to growth factors, insulin, or other pathway activators

  • Use time-course experiments to establish the sequence of events in pathway activation

  • Examine how pathway inhibitors affect PIK3R3 expression and function

• Cancer-specific considerations:

  • In liver cancer models, PIK3R3 has been shown to activate Akt signaling and regulate the expression of downstream targets like CDKN1C and SMC1A

  • Knockdown of PIK3R3 impairs tumor cell growth, while overexpression enhances proliferation

  • These effects appear to be mediated through control of cell cycle progression

• Feedback regulation:

  • Investigate potential feedback loops within the pathway that may affect PIK3R3 expression or function

  • Consider how other PI3K regulatory subunits might compensate for PIK3R3 modulation

  • Examine cross-talk with other signaling pathways that may influence PI3K/Akt signaling

• Therapeutic implications:

  • Assess how PI3K/Akt pathway inhibitors affect PIK3R3 expression and function

  • Investigate whether PIK3R3 status affects sensitivity to pathway-targeted therapies

  • Consider PIK3R3 as a potential biomarker for predicting treatment response

Research has demonstrated that PIK3R3-activated Akt signaling determines the expression of cell cycle regulators like CDKN1C in liver cancer cells, establishing a mechanistic link between PIK3R3 overexpression and enhanced tumor growth .

What are common technical challenges when using PIK3R3 antibodies and how can they be addressed?

Researchers may encounter several technical challenges when working with PIK3R3 antibodies. Here are common issues and their solutions:

• Weak or absent signal:

  • Increase antibody concentration or incubation time

  • Enhance antigen retrieval for fixed tissues (extend time or try alternative buffers)

  • Use more sensitive detection systems (amplified polymer systems for IHC, enhanced chemiluminescence for WB)

  • Ensure sample preparation preserves protein integrity (use fresh protease inhibitors)

  • Verify PIK3R3 expression level in your sample type (use positive control tissues like brain or testis)

• Multiple bands in Western blotting:

  • PIK3R3 has a calculated molecular weight of 54.4 kDa

  • Additional bands may represent splice variants, degradation products, or cross-reactivity

  • Validate specificity through knockdown experiments

  • Use gradient gels (4-15%) for better resolution

  • Consider antibodies targeting different epitopes to confirm findings

• High background in immunostaining:

  • Optimize blocking (use 5-10% serum from secondary antibody species)

  • Increase washing duration and frequency

  • Reduce primary and secondary antibody concentrations

  • For IHC, ensure complete deparaffinization and proper blocking of endogenous peroxidase

  • For IF, include background-reducing reagents or consider Sudan Black B treatment

• Inconsistent results between experiments:

  • Standardize sample collection and processing protocols

  • Use consistent antibody lots when possible

  • Include reference standards across experiments

  • Maintain detailed records of all experimental conditions

  • Process control and experimental samples simultaneously

• Discrepancies between antibody reactivity and RNA expression:

  • Consider post-transcriptional regulation affecting protein levels

  • Verify antibody specificity through additional validation approaches

  • Examine potential technical issues with either RNA or protein detection methods

  • Consider temporal differences in mRNA versus protein expression

How should I interpret different patterns of PIK3R3 expression in experimental and clinical samples?

Interpreting PIK3R3 expression patterns requires careful consideration of biological context and technical factors:

• Baseline tissue expression:

  • Highest expression normally occurs in brain and testis tissues

  • Moderate expression in adipose tissue, kidney, heart, lung, and skeletal muscle

  • Low expression in other tissues should be interpreted cautiously and verified with appropriate controls

• Cancer-specific expression patterns:

  • Significant upregulation has been documented in liver cancer tissues compared to normal liver

  • PIK3R3 is also upregulated in ovarian cancer and correlates with prognosis

  • Expression is elevated in cancer stem cell populations, including spheroid cultures of ovarian cancer cells

  • PIK3R3 levels are increased in high-grade serous ovarian cancer (HGSOC) tumor organoids compared to fallopian tube normal organoids

• Subcellular localization considerations:

  • PIK3R3 typically functions in the cytoplasm as part of PI3K complexes

  • Changes in localization patterns may indicate altered function

  • Compare distribution patterns between normal and pathological samples

  • Correlate localization with activation status of the PI3K/Akt pathway

• Expression heterogeneity:

  • Account for cellular heterogeneity within tissues and tumors

  • Consider regional variation in expression, particularly in tumor samples

  • Correlate with markers of cell type, differentiation status, or stemness

• Analytical approaches:

  • Use quantitative methods when possible (digital image analysis for IHC, densitometry for WB)

  • Compare expression relative to appropriate control samples

  • Consider both intensity and distribution patterns in tissue sections

  • Correlate with clinical parameters when analyzing patient samples

Research has demonstrated that PIK3R3 expression correlates with proliferation markers and influences cell cycle distribution, with overexpression promoting G2M distribution compared to control groups .

How can I quantitatively analyze PIK3R3 expression changes in response to experimental manipulations?

Accurate quantification of PIK3R3 expression changes requires rigorous methodology:

• Western blot quantification:

  • Use digital imaging systems with linear dynamic range

  • Include loading controls (β-actin, GAPDH) or total protein normalization

  • Apply appropriate background subtraction

  • Calculate relative expression using integrated density values

  • Include biological replicates (minimum n=3) and appropriate statistical analysis

  • Present data with both representative images and quantification graphs

• Immunohistochemistry quantification:

  • Use standardized scoring systems (H-score, Allred score) or digital image analysis

  • Score multiple fields per sample to account for heterogeneity

  • Consider both staining intensity and percentage of positive cells

  • Implement blinded assessment when possible

  • Validate quantification with alternative methods

• qRT-PCR for complementary analysis:

  • Use validated PIK3R3-specific primers

  • Include multiple reference genes for normalization

  • Apply the 2^(-ΔΔCt) method for relative quantification

  • Correlate mRNA and protein expression changes

• Analyzing functional consequences:

  • Link PIK3R3 expression changes to functional outcomes

  • For cancer studies, correlate with proliferation markers (Ki67, EdU incorporation)

  • Assess effects on cell cycle distribution using flow cytometry

  • Examine colony formation capacity as demonstrated in liver cancer studies

• Time-course and dose-response studies:

  • Design experiments to capture dynamic changes

  • Include appropriate time points based on expected response kinetics

  • For treatment studies, use multiple concentrations to establish dose-dependency

  • Apply appropriate statistical methods for time-series data

Research on liver cancer has demonstrated that PIK3R3 overexpression significantly improves tumor volume compared to controls in vivo, with quantitative analysis showing progressive divergence in tumor size over time . Similarly, colony formation assays and EdU incorporation studies have quantitatively demonstrated enhanced cell growth upon PIK3R3 overexpression .

What emerging applications for PIK3R3 antibodies should researchers consider exploring?

As our understanding of PIK3R3 biology expands, several promising research directions emerge:

• Single-cell analysis of PIK3R3 expression:

  • Apply PIK3R3 antibodies in single-cell protein analysis technologies

  • Investigate cellular heterogeneity within tumors and normal tissues

  • Correlate PIK3R3 expression with cell states and differentiation status

  • Combine with other markers to identify PIK3R3-high subpopulations

• Therapeutic response biomarkers:

  • Evaluate PIK3R3 as a predictive biomarker for PI3K/Akt pathway inhibitors

  • Determine whether PIK3R3 expression levels correlate with treatment outcomes

  • Develop standardized IHC protocols for potential clinical application

  • Investigate PIK3R3 expression changes during treatment as pharmacodynamic markers

• Combination with emerging technologies:

  • Apply multiplexed immunofluorescence to study PIK3R3 in complex tissue microenvironments

  • Utilize spatial transcriptomics alongside protein detection for integrated analysis

  • Implement mass cytometry (CyTOF) for high-dimensional analysis of PIK3R3 in relation to numerous other markers

  • Develop proximity-based assays to study PIK3R3 protein interactions in situ

• Cancer stemness and resistance mechanisms:

  • Further investigate the elevated PIK3R3 expression observed in cancer stem cells

  • Examine the relationship between PIK3R3 and therapy resistance

  • Study PIK3R3's role in tumor initiation and metastasis

  • Develop combinatorial approaches targeting PIK3R3-dependent pathways

• Expanding beyond cancer:

  • Investigate PIK3R3's role in metabolic disorders, given its interaction with insulin signaling components

  • Explore potential functions in neurological conditions, considering its high expression in brain tissue

  • Examine developmental roles using spatiotemporal expression analysis

  • Consider potential involvement in inflammatory and immune processes

Current research has established PIK3R3's importance in liver and ovarian cancers , but its role in other malignancies and physiological processes remains to be fully elucidated, presenting numerous opportunities for future investigation.