Phospho-AKT1 (Ser473) Recombinant Monoclonal Antibody

What is the Phospho-AKT1 (Ser473) antibody and what does it specifically detect?

The Phospho-AKT1 (Ser473) antibody is a monoclonal antibody specifically designed to detect the AKT1 protein only when phosphorylated at the Serine 473 residue. This post-translational modification is critical for full activation of AKT1 kinase function. The antibody binds exclusively to the phosphorylated form of this specific serine residue, making it an essential tool for monitoring AKT activation states in various experimental contexts . The specificity of the antibody is typically established through immunization with synthetic peptides derived from human phospho-AKT1 (Ser473) regions, followed by careful selection and cloning of B cells that produce antibodies with high specificity for this epitope .

How is the Phospho-AKT1 (Ser473) recombinant monoclonal antibody produced?

The production process involves a sophisticated combination of protein technology and DNA recombinant techniques. Initially, mice are immunized with synthetic peptides derived from human phospho-AKT1 (Ser473), triggering B cell production against this specific epitope. Positive B cells that produce antibodies with the desired specificity are isolated and undergo single clone identification. The genes encoding these antibodies are then amplified using PCR and inserted into plasmid vectors to create recombinant vectors. These vectors are subsequently introduced into host cells that express the recombinant antibody. The final product is purified from the cell culture supernatant using affinity chromatography to ensure high purity and consistency . This recombinant approach offers superior batch-to-batch consistency compared to traditional hybridoma-based monoclonal antibody production methods.

What is the biological significance of AKT1 phosphorylation at Serine 473?

Phosphorylation of AKT1 at Serine 473 represents a crucial regulatory mechanism in the PI3K/AKT signaling pathway. This specific phosphorylation event is essential for complete activation of AKT1's kinase function and occurs downstream of PI3K activation. AKT1 plays fundamental roles in controlling multiple cellular processes including apoptosis, cell proliferation, transcription, cell migration, and glucose metabolism . The phosphorylation at Ser473 is particularly important for cell survival mechanisms, making it a significant marker in cancer research . In normal cells, this phosphorylation is tightly regulated, but in many cancer types, constitutive phosphorylation drives oncogenic processes, highlighting the importance of reliable detection methods for this modification .

What are the subcellular localization patterns of phosphorylated AKT1?

Phosphorylated AKT1 exhibits dynamic localization patterns that reflect its activation state and functional roles. The protein can be found in multiple cellular compartments including the cytoplasm, nucleus, and cell membrane . Upon activation through phosphorylation, AKT1 can translocate to the nucleus, a process enhanced by interaction with TCL1A. Additionally, phosphorylation on Tyr-176 by TNK2 facilitates localization to the cell membrane, where AKT1 becomes accessible for further phosphorylation at Thr-308 and Ser-473, leading to full activation . This activated form subsequently translocates to the nucleus to regulate transcription factors involved in survival and proliferation pathways. The subcellular distribution pattern serves as an important indicator of AKT1 activation status and can be visualized using immunofluorescence techniques with phospho-specific antibodies .

What are the recommended applications for Phospho-AKT1 (Ser473) antibodies in research?

Phospho-AKT1 (Ser473) antibodies are versatile tools with validated applications across multiple experimental platforms. Based on extensive testing, these antibodies are recommended for:

• Western Blot (WB): Using dilutions ranging from 1:500 to 1:5000, with optimal results typically at 1:1000-2000

• Immunohistochemistry (IHC): Effective at dilutions of 1:50 to 1:200, particularly for paraffin-embedded sections

• Immunofluorescence (IF): Functional at 1:20 to 1:200 dilutions for cellular localization studies

• Immunoprecipitation (IP): Recommended at 1:200 to 1:1000 dilutions

• Flow Cytometry (FC): Validated for intracellular staining applications

• ELISA: Suitable for quantitative detection in plate-based formats

• HTRF (Homogeneous Time-Resolved Fluorescence): Specialized application for high-throughput screening

Each application requires specific optimization based on sample type, fixation method, and detection system, but these recommended ranges provide a reliable starting point for experimental design .

How can HTRF technology be used to detect Phospho-AKT1 (Ser473) in high-throughput screening?

HTRF (Homogeneous Time-Resolved Fluorescence) technology offers a sophisticated plate-based approach for quantifying AKT1 phosphorylation at Ser473, particularly valuable for high-throughput screening applications. Unlike Western blotting, HTRF eliminates the need for gels, electrophoresis, or transfer steps, streamlining the workflow in a no-wash format.

The assay employs two labeled antibodies: one with a donor fluorophore specifically binding to the phosphorylated Ser473 motif, and another with an acceptor fluorophore recognizing AKT1 independent of its phosphorylation state. When AKT1 is phosphorylated, these antibodies form an immune complex bringing the donor and acceptor fluorophores into close proximity, generating a FRET signal proportional to the concentration of phosphorylated protein .

The HTRF protocol can be performed in either a two-plate format (cells cultured, lysed, and transferred to detection plates) or a single-plate format optimized for HTS applications. Sample volumes are typically 16 μL, with assay kits designed for 500 data points. For validation, specific blocking peptides can be added at different concentrations before detection reagents to confirm signal specificity. After overnight incubation at room temperature, the FRET signal is measured, providing quantitative readout of AKT1 phosphorylation levels . This approach is particularly valuable for drug screening aimed at PI3K pathway modulation.

What are the critical considerations for Western blot detection of Phospho-AKT1 (Ser473)?

Western blot detection of Phospho-AKT1 (Ser473) requires specific technical considerations to ensure reliable results:

• Sample Preparation: Samples must be collected with phosphatase inhibitors to prevent dephosphorylation during processing. Flash-freezing samples immediately after collection helps preserve phosphorylation status .

• Expected Molecular Weight: While the theoretical molecular weight of AKT1 is around 56 kDa, the phosphorylated form typically migrates at 60-62 kDa on SDS-PAGE gels due to the impact of phosphorylation on protein mobility . Researchers should be aware that mobility rates can be affected by other modifications, potentially resulting in multiple bands that don't align precisely with expected sizes .

• Antibody Dilution: Optimal dilutions range from 1:500 to 1:5000, with most applications finding 1:1000-2000 to be effective . Titration is recommended for each new lot of antibody.

• Blocking Conditions: 5% BSA in TBST is typically more effective than milk-based blockers, as milk contains phosphatases that may reduce signal .

• Positive Controls: Lysates from IGF-1 stimulated cells (such as SH-SY5Y) provide reliable positive controls, as IGF-1 activates the PI3K/AKT pathway leading to increased Ser473 phosphorylation .

• Stripping and Reprobing: If total AKT detection is required after phospho-detection, mild stripping conditions are recommended to avoid epitope damage .

These considerations help ensure specific detection of the phosphorylated form and minimize false negative or positive results in experimental settings.

What sample types and species reactivity have been validated for Phospho-AKT1 (Ser473) antibodies?

Phospho-AKT1 (Ser473) antibodies have undergone extensive cross-species validation across various sample types. The primary validated species reactivity includes:

• Human: Consistently demonstrated reactivity across numerous cell lines and tissue samples, including cancer cell lines like U-87 MG and SH-SY5Y

• Mouse: Validated in multiple studies with strong cross-reactivity

• Rat: Confirmed reactivity in various experimental systems

Additional species with reported reactivity in published studies include:

• Pig

• Rabbit

• Canine

• Non-human primates

• Chicken

• Zebrafish

• Duck

The antibodies have been successfully applied to various sample types:

• Cell lysates (from cultured cell lines)

• Tissue sections (both frozen and paraffin-embedded)

• Primary cell isolates

For tissue-specific applications, the antibody has shown particularly strong performance in:

• Cancer tissues (lung carcinoma specifically mentioned)

• Neuronal cells (SH-SY5Y)

• Glioblastoma (U-87 MG)

This broad cross-species reactivity makes these antibodies versatile tools for comparative studies across different model organisms and diverse experimental conditions .

How can Phospho-AKT1 (Ser473) antibodies be used to study PI3K pathway activation in cancer models?

Phospho-AKT1 (Ser473) antibodies serve as critical tools for monitoring PI3K pathway activation in cancer research through multiple sophisticated approaches:

• Monitoring Treatment Response: The antibodies can quantify changes in AKT phosphorylation following treatment with PI3K/AKT/mTOR pathway inhibitors, providing direct evidence of target engagement and pathway modulation . This application is particularly valuable for drug development and resistance studies.

• Cancer Progression Analysis: Studies have revealed that Tyr-176 phosphorylated forms of AKT1 show significant increases during cancer progression from normal tissue through hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), to lymph node metastatic (LNMM) stages . Using phospho-AKT1 antibodies enables quantification of these progressive changes.

• Multiplex Signaling Analysis: Combined with other phospho-specific antibodies, researchers can map complex signaling networks by simultaneously measuring multiple phosphorylation events in response to stimuli or inhibitors. HTRF-based platforms are particularly suitable for this application .

• Patient-Derived Xenograft Models: The antibodies' cross-species reactivity makes them suitable for studies using patient-derived samples in animal models, enabling translational research that bridges clinical and laboratory investigations .

• Immunofluorescence Co-localization: Advanced imaging using phospho-AKT1 antibodies can reveal spatial relationships between activated AKT1 and other signaling components, providing insights into compartmentalized signaling mechanisms .

These applications collectively contribute to understanding the role of AKT activation in cancer initiation, progression, and therapeutic resistance mechanisms.

What are the recommended approaches for studying phosphorylation-dependent protein interactions of AKT1?

Studying phosphorylation-dependent protein interactions of AKT1 requires specialized methodological approaches:

• Co-Immunoprecipitation with Phospho-Specific Antibodies: Phospho-AKT1 (Ser473) antibodies can be used at 1:200-1:1000 dilutions for immunoprecipitation, allowing selective enrichment of the phosphorylated form and its interacting partners . This approach helps identify proteins that specifically interact with the activated form of AKT1.

• Proximity Ligation Assays (PLA): This technique combines phospho-AKT1 antibodies with antibodies against putative interaction partners, generating fluorescent signals only when proteins are in close proximity (<40 nm), providing in situ visualization of interactions specifically involving phosphorylated AKT1.

• Phospho-Proteomic Mass Spectrometry: Coupling immunoprecipitation using phospho-AKT1 antibodies with mass spectrometry analysis can identify both the interactome and phosphorylation status of interaction partners, revealing complex signaling networks.

• HTRF-Based Interaction Assays: The HTRF technology can be adapted to study direct protein-protein interactions by labeling potential binding partners with compatible fluorophores and measuring interaction-dependent FRET signals .

• Phosphorylation-Dependent Subcellular Localization: Immunofluorescence studies using phospho-AKT1 antibodies can track how phosphorylation affects interactions with nuclear transport machinery, revealing mechanisms behind the observed nuclear translocation enhanced by TCL1A interaction .

These approaches provide complementary information about how Ser473 phosphorylation modulates AKT1's interaction landscape, helping decode its role in diverse cellular processes.

How can phosphorylation dynamics of AKT1 be monitored in live cells?

Monitoring phosphorylation dynamics of AKT1 in live cells requires approaches that complement traditional fixed-cell antibody-based methods:

• Genetically Encoded FRET-Based Biosensors: These constructs contain the AKT1 phosphorylation motif flanked by fluorescent proteins that undergo FRET changes upon phosphorylation. While not directly using antibodies, these systems can be validated using phospho-AKT1 (Ser473) antibodies in parallel fixed-cell experiments .

• Phospho-Flow Cytometry: For suspension cells or cells that can be detached without compromising signaling, intracellular staining with phospho-AKT1 antibodies followed by flow cytometry allows quantitative assessment of phosphorylation across populations with single-cell resolution .

• Time-Course Fixation Studies: While not truly "live," preparing multiple samples fixed at specific timepoints after stimulation allows temporal resolution of phosphorylation events. Combining immunofluorescence with phospho-AKT1 antibodies and high-content imaging enables quantitative spatiotemporal analysis .

• Cell-Permeable Phospho-Sensors: Peptide-based sensors that change fluorescence properties upon binding to phospho-motifs can be delivered to live cells, though their specificity should be validated against antibody-based methods.

• Correlative Live-Cell/Fixed-Cell Imaging: Cells expressing fluorescently tagged AKT1 can be imaged live to track localization, then fixed and stained with phospho-specific antibodies to correlate localization with phosphorylation status.

These approaches provide complementary information about the temporal and spatial dynamics of AKT1 phosphorylation events that cannot be captured by static antibody-based detection alone.

What is the relationship between AKT1 phosphorylation at Ser473 and other post-translational modifications?

AKT1 undergoes multiple post-translational modifications that work in concert to regulate its activity and function:

• Coordinated Phosphorylation Events: Phosphorylation at Ser473 works cooperatively with phosphorylation at Thr308. While Thr308 phosphorylation (by PDK1) is necessary for partial activation, Ser473 phosphorylation (by mTORC2) is required for full kinase activity. Both modifications should be monitored for comprehensive understanding of activation status .

• Hierarchical Phosphorylation Sequence: Phosphorylation at Tyr176 by TNK2 facilitates membrane localization, which is a prerequisite for subsequent phosphorylation at Thr308 and Ser473. This creates a sequential phosphorylation cascade that can be monitored using site-specific antibodies .

• Cross-talk with Ubiquitination: Ubiquitination of AKT1 can affect both its stability and activation state. Phosphorylation at Ser473 can influence ubiquitination patterns by altering protein conformation and accessibility of lysine residues to ubiquitin ligases.

• Acetylation Interactions: AKT1 can also undergo acetylation, which may compete with phosphorylation for certain residues or alter the accessibility of phosphorylation sites.

• SUMOylation Effects: SUMOylation of AKT1 can affect its nuclear localization and activity, potentially interacting with the phosphorylation-dependent nuclear translocation mechanisms enhanced by TCL1A .

Understanding these interrelationships requires multiparametric analysis, combining phospho-specific antibodies with detection methods for other modifications to build comprehensive models of AKT1 regulation.

Why might the observed molecular weight of phosphorylated AKT1 differ from the expected molecular weight?

The discrepancy between observed and expected molecular weights of phosphorylated AKT1 is a common technical issue with several possible explanations:

• Effect of Phosphorylation on Mobility: Phosphorylation adds negative charges that can affect protein migration in SDS-PAGE. While the theoretical molecular weight of AKT1 is approximately 56 kDa, the phosphorylated form typically migrates at 60-62 kDa due to these conformational changes .

• Multiple Modification States: AKT1 can harbor various post-translational modifications simultaneously (phosphorylation at multiple sites, ubiquitination, acetylation). This can result in multiple bands representing different modification states of the protein .

• Protein Isoforms: Though antibodies may target AKT1 specifically, cross-reactivity with closely related isoforms (AKT2, AKT3) that share high sequence homology but differ slightly in molecular weight can occur.

• Proteolytic Processing: Incomplete inhibition of proteases during sample preparation can lead to partial degradation, resulting in lower molecular weight bands.

• Experimental Conditions: Gel concentration, running buffer composition, and voltage can all affect protein migration patterns and apparent molecular weight.

Researchers should validate the specificity of bands using positive controls, blocking peptides, and when possible, AKT1 knockout/knockdown samples. As noted in product documentation: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size" .

What are the optimal cell stimulation conditions for studying AKT1 phosphorylation at Ser473?

Optimizing cell stimulation conditions is crucial for robust phospho-AKT1 (Ser473) detection:

• Growth Factor Stimulation:

  • IGF-1: Human IGF-1 is highly effective, with 15-minute stimulation protocols demonstrated for SH-SY5Y cells

  • Insulin: 10-100 nM for 5-30 minutes in insulin-responsive cell lines

  • EGF: 10-50 ng/mL for 5-15 minutes in epithelial or cancer cell lines

  • PDGF: Particularly effective in fibroblast and smooth muscle cell models

• Serum Starvation: Prior serum starvation (typically 4-24 hours) significantly enhances the phosphorylation response by reducing baseline activation . This creates a cleaner experimental system with lower background.

• Timing Considerations: Phosphorylation at Ser473 is typically rapid, peaking between 5-30 minutes after stimulation depending on the stimulus and cell type. Time-course experiments are recommended for new systems .

• Inhibitor Studies: For pathway validation, pre-treatment with PI3K inhibitors (LY294002, Wortmannin) or mTOR inhibitors (rapamycin, Torin1) can confirm the specificity of the phosphorylation event.

• Sample Processing: Immediate lysis in buffer containing phosphatase inhibitors is critical, as phosphorylation states can change rapidly during sample processing .

These conditions have been validated across multiple experimental systems and provide a strong foundation for studying AKT1 phosphorylation dynamics in diverse research contexts.

How can specificity of phospho-AKT1 (Ser473) antibody signals be validated?

Rigorous validation of phospho-AKT1 (Ser473) antibody specificity involves multiple complementary approaches:

• Blocking Peptide Competition: Adding increasing concentrations of phospho-peptides used as immunogens can competitively inhibit antibody binding. In HTRF assays, this approach has been demonstrated with blocking peptides specific to AKT1, confirming signal specificity in a dose-dependent manner .

• Phosphatase Treatment Controls: Treating positive control samples with lambda phosphatase to remove phosphorylation should eliminate signal from true phospho-specific antibodies.

• Kinase Inhibitor Studies: Pre-treatment of cells with specific inhibitors of the PI3K/AKT/mTOR pathway (such as PI3K inhibitors LY294002/Wortmannin or mTORC2 inhibitors) should reduce signal in proportion to their effectiveness at blocking Ser473 phosphorylation.

• Knockout/Knockdown Validation: Using AKT1 knockout or knockdown models provides definitive evidence of antibody specificity. Published studies using this approach are available as reference points .

• Parallel Detection Methods: Comparing results across different detection platforms (Western blot, ELISA, immunofluorescence) with the same antibody can reveal platform-specific artifacts.

• Isoform Specificity Testing: Testing against AKT2 and AKT3 phosphorylated at equivalent serine residues to confirm AKT1 specificity when isoform-specific detection is critical.

These validation approaches should be applied systematically when working with new experimental systems or antibody lots to ensure reliable interpretation of results.

What are the key considerations for quantitative analysis of AKT1 phosphorylation in tissue samples?

Quantitative analysis of AKT1 phosphorylation in tissue samples presents unique challenges requiring specialized approaches:

• Tissue Collection and Fixation: Phosphorylation states can change rapidly post-mortem. Immediate fixation or flash-freezing is essential, with phosphatase inhibitors incorporated at all stages. For IHC applications, optimal fixation times should be determined empirically, as overfixation can mask phospho-epitopes .

• Antigen Retrieval Optimization: Phospho-epitopes often require more aggressive antigen retrieval methods than total protein detection. For AKT1 phospho-Ser473, citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with heat-induced epitope retrieval have proven effective for paraffin-embedded samples .

• Signal Normalization Strategies:

  • Serial sections stained for total AKT1

  • Duplex immunofluorescence for simultaneous detection of phospho and total AKT1

  • Normalization to housekeeping proteins stable in the tissue/disease context

• Tissue-Specific Considerations: Different tissues exhibit varying baseline phosphorylation levels. For example, brain tissues typically show higher constitutive AKT phosphorylation than many other tissues .

• Quantification Methods:

  • H-score system (combining intensity and percentage of positive cells)

  • Digital image analysis with validated algorithms

  • Phospho/total ratios when dual staining is possible

• Controls for Tissue Analysis:

  • Known positive tissues (e.g., human lung carcinoma has been validated)

  • Adjacent normal tissue for internal comparison

  • Phosphatase-treated serial sections as negative controls

These considerations help ensure that quantitative differences in phosphorylation status reflect biological reality rather than technical artifacts in complex tissue environments.

How are new generations of phospho-AKT1 antibodies being developed to enhance detection sensitivity and specificity?

The development of next-generation phospho-AKT1 antibodies focuses on multiple technological advancements:

• Recombinant Antibody Technology: The transition from hybridoma-derived to recombinant antibodies represents a significant advancement, offering superior batch-to-batch consistency through precise genetic control of antibody production . This approach eliminates the variability inherent in traditional monoclonal antibody production.

• Single B Cell Cloning Approaches: Advanced methods for selecting and immortalizing individual B cells with exceptional specificity for phospho-epitopes are enhancing the precision of antibody development .

• Affinity Maturation Techniques: In vitro directed evolution and computational design approaches are being applied to optimize binding characteristics, increasing both affinity and specificity for the phospho-Ser473 epitope.

• Fragment-Based Antibodies: Development of smaller antibody formats (nanobodies, single-chain variable fragments) that may access phospho-epitopes more effectively in certain applications, particularly for intracellular targets or sterically hindered phosphorylation sites.

• Multiparametric Detection Formats: Creation of bispecific or multispecific antibodies that can simultaneously detect both phosphorylated Ser473 and other modifications or total AKT1, enabling direct ratio measurements within a single detection system .

These advancements promise to enhance the utility of phospho-AKT1 antibodies across research applications, potentially enabling detection of lower abundance phosphorylation events and more precise quantification in complex biological samples.

What emerging technologies are integrating phospho-AKT1 detection for high-throughput drug discovery?

Emerging technologies are revolutionizing phospho-AKT1 detection in drug discovery pipelines:

• HTRF-Based Plate Assays: These homogeneous, no-wash formats have transformed high-throughput screening by eliminating labor-intensive steps like gels and transfers. The approach uses a dual-antibody system with donor and acceptor fluorophores to generate FRET signals proportional to phosphorylation levels . This technology is particularly valuable for screening compound libraries affecting the PI3K/AKT pathway.

• Automated Western Blot Systems: Platforms incorporating automated sample preparation, electrophoresis, and detection have increased throughput and reproducibility of traditional Western blot approaches for phospho-AKT1 detection.

• Microfluidic Immunoassays: These systems require minimal sample volumes (nanoliters) and can deliver quantitative phospho-AKT1 measurements from limited material, enabling screening with patient-derived samples or rare cell populations.

• High-Content Imaging Platforms: Automated microscopy systems with machine learning-based image analysis can quantify phospho-AKT1 levels in individual cells within heterogeneous populations, providing single-cell resolution data critical for understanding cellular heterogeneity in drug response.

• Bead-Based Multiplex Assays: Technologies allowing simultaneous measurement of multiple phosphorylation events (including AKT1-Ser473) facilitate comprehensive pathway analysis from single samples, critical for understanding complex signaling network responses to drug candidates.