AKT3 Antibody, FITC conjugated

What is AKT3 and why is it important for research?

AKT3, also known as protein kinase Bγ (PKBγ), is the third member of the protein kinase B family. It is predominantly expressed in the brain, testis, and certain cancer cell types. AKT3 functions similarly to other AKT isoforms by being recruited to the cell membrane upon binding to PIP3 generated through PI3K activation, followed by phosphorylation by upstream kinases such as PDK1 . The activated AKT3 subsequently phosphorylates various substrates including BAD proteins that promote cell survival, FOXO transcription factors, and components of the mTOR signaling pathway . Its critical role in neural development, synaptic plasticity, and cognitive functions makes it an important target for neuroscience research . Aberrant expression or dysfunction of AKT3 has been associated with various diseases including neurodegenerative disorders, psychiatric conditions, and certain cancers, making it a valuable research target .

What are FITC-conjugated AKT3 antibodies and how do they work?

FITC-conjugated AKT3 antibodies are immunological reagents in which an anti-AKT3 antibody is chemically linked to fluorescein isothiocyanate (FITC), a fluorescent dye that emits green light when excited at approximately 490nm, with emission at around 525nm . These conjugated antibodies enable direct visualization of AKT3 protein in various experimental contexts without requiring secondary antibody labeling steps. The antibody portion specifically binds to AKT3 protein (either phosphorylated, unphosphorylated, or both forms, depending on the antibody's epitope specificity), while the FITC component provides the fluorescent signal for detection . Available as both mouse monoclonal (such as clone 25F6.F6.D8) and rabbit monoclonal (such as clone K17-H) formats, these antibodies can be used to detect human, mouse, and rat AKT3, depending on the specific product .

What applications are FITC-conjugated AKT3 antibodies suitable for?

FITC-conjugated AKT3 antibodies are particularly well-suited for flow cytometry applications, where they allow detection of AKT3 expression in individual cells within heterogeneous populations . The available products have been validated for:

When using these antibodies for immunohistochemistry, it's important to note that they work best with fresh frozen tissues, as attempts to stain paraffin-embedded formalin-fixed tissues have yielded negative results .

How can researchers optimize the use of AKT3 FITC antibodies in flow cytometry?

For optimal flow cytometry results with AKT3 FITC antibodies, researchers should implement the following methodological approaches:

• Cell preparation: Proper fixation and permeabilization are critical since AKT3 is primarily an intracellular target. Use fixation/permeabilization buffers compatible with phospho-epitope preservation if detecting phosphorylated forms .

• Antibody dilution: Follow manufacturer-recommended dilutions, typically 10 μl per test for antibodies like the K17-H clone . Titration experiments may be necessary to determine optimal concentration for specific cell types.

• Controls: Always include appropriate isotype controls to distinguish true signals from background fluorescence. Flow cytometry validation data shows clear separation between AKT3-stained samples (red profile) and isotype controls (black profile) in human blood lymphocytes from chronic lymphocytic leukemia patients .

• Compensation: When performing multi-color flow cytometry, proper compensation is essential due to FITC's emission spectrum overlap with other fluorophores like PE.

• Instrument settings: The optimal excitation for FITC is 490nm with emission detected at 525nm. Ensure proper instrument calibration before analysis .

• Sample storage: Protect stained samples from light exposure to prevent photobleaching of the FITC fluorophore, and analyze samples promptly for best results .

What approaches can distinguish AKT3 from other AKT isoforms in experimental systems?

Distinguishing AKT3 from other AKT isoforms (AKT1 and AKT2) presents a significant challenge due to their high sequence homology. Researchers can employ several strategies:

What role does AKT3 play in neuroinflammatory conditions and how can FITC-conjugated antibodies help investigate this?

AKT3 plays a crucial neuroprotective role during inflammatory demyelinating diseases as demonstrated in experimental autoimmune encephalomyelitis (EAE) models. Key findings include:

• Protection against demyelination: Akt3−/− mice exhibit worse clinical outcomes during myelin-oligodendrocyte glycoprotein (MOG)-induced EAE, with severe demyelination and increased inflammation compared to wild-type controls .

• T-cell-specific effects: Mice with enhanced Akt3 kinase activity (Akt3Nmf350) demonstrate lower clinical scores, delayed disease onset, reduced inflammatory cell infiltration, and less axonal damage during EAE .

• Regulatory T-cell differentiation: Akt3Nmf350 mice show increased efficiency in differentiation toward FOXP3-expressing induced regulatory T cells (iTregs), while conditional deletion of Akt3 in CD4+ T cells results in earlier EAE onset and reduced FOXP3+ cells .

• Cell-specific importance: Conditional deletion of Akt3 in neurons (Syn1-CKO) showed no difference in EAE outcome, highlighting that Akt3's protective effects during inflammatory demyelination are primarily mediated through T-cells rather than neurons .

FITC-conjugated AKT3 antibodies can contribute to investigating these processes by:

• Tracking AKT3 expression in different immune cell populations via flow cytometry during disease progression

• Monitoring changes in AKT3 levels in response to therapeutic interventions

• Identifying AKT3-expressing cells in tissue sections from neuroinflammatory lesions

• Correlating AKT3 expression with markers of regulatory T-cell function (e.g., FOXP3)

What experimental controls should be included when using AKT3 FITC antibodies?

Proper experimental controls are essential for generating reliable data with AKT3 FITC antibodies:

Flow cytometry validation data shows clear differentiation between AKT3-stained samples and isotype controls in human blood lymphocytes from chronic lymphocytic leukemia patients, demonstrating the importance of proper control implementation .

How can AKT3 FITC antibodies contribute to understanding T-cell-mediated neuroprotection?

Recent research has established that Akt3 signaling in T-cells plays a critical role in maintaining CNS integrity during inflammatory demyelinating diseases . AKT3 FITC antibodies can be instrumental in advancing this research through several approaches:

• Tregs quantification and characterization: Flow cytometric analysis using AKT3 FITC antibodies in combination with FOXP3 markers can help quantify and characterize regulatory T-cell populations in different experimental conditions. This is particularly relevant given that Akt3Nmf350 mice show increased efficiency in differentiation toward FOXP3-expressing induced regulatory T cells .

• Signaling pathway analysis: By combining AKT3 FITC antibodies with phospho-specific antibodies against downstream targets, researchers can map signaling cascades in T-cells during neuroinflammatory conditions.

• Cell sorting applications: FITC-conjugated AKT3 antibodies enable isolation of AKT3-expressing T-cell subpopulations for further functional studies or transcriptomic analysis.

• In vivo tracking: Adoptive transfer experiments with labeled T-cells can be analyzed using AKT3 FITC antibodies to track cell migration and function in neuroinflammatory contexts.

• Therapeutic intervention assessment: These antibodies can help evaluate how potential therapeutic agents affect AKT3 expression and activity in T-cells during treatment of inflammatory demyelinating conditions.

The experimental evidence showing that Akt3−/− mice have significantly worse clinical courses during EAE, together with data demonstrating that conditional deletion of Akt3 in CD4+ T-cells results in earlier disease onset, highlights the importance of T-cell AKT3 in neuroprotection .

What methodological approaches are recommended for detecting both phosphorylated and unphosphorylated forms of AKT3?

Some AKT3 antibodies, including the FITC-conjugated mouse monoclonal anti-AKT3 (clone 25F6.F6.D8), are capable of detecting both phosphorylated and unphosphorylated forms of the protein . To optimize detection and differentiation between these forms:

• Fixation considerations: Use phospho-preserving fixatives such as paraformaldehyde followed by methanol post-fixation to maintain phospho-epitopes.

• Dual staining approach: Combine the AKT3 FITC antibody that detects total AKT3 with a phospho-specific antibody conjugated to a different fluorophore to simultaneously visualize total and phosphorylated protein.

• Phosphatase treatment controls: Include phosphatase-treated samples as controls to confirm phosphorylation-specific detection.

• Stimulation experiments: Compare AKT3 detection in resting cells versus those stimulated with growth factors that activate the PI3K pathway to validate phosphorylation-responsive detection.

• Western blot validation: Confirm flow cytometry results with western blotting using phospho-specific antibodies, expecting a band of approximately 56 kDa corresponding to AKT3 protein .

What are the technical considerations for immunohistochemical applications of AKT3 FITC antibodies?

When utilizing AKT3 FITC antibodies for immunohistochemistry, several technical factors should be considered:

• Tissue preparation: Fresh frozen tissues are strongly recommended as attempts at staining paraffin-embedded formalin-fixed tissues have yielded negative results with these antibodies .

• Pre-treatment requirements: No pre-treatment of samples is generally required when using fresh frozen tissues, simplifying the protocol .

• Autofluorescence management: Brain tissue often exhibits high autofluorescence, which can interfere with FITC signal detection. Consider using Sudan Black B or similar treatments to reduce autofluorescence.

• Signal amplification: For weaker signals, consider using anti-FITC secondary antibodies conjugated to brighter fluorophores or enzymatic detection systems.

• Co-localization studies: When performing double or triple immunofluorescence, select companion fluorophores with minimal spectral overlap with FITC to avoid bleed-through.

• Storage of slides: Mounted slides should be stored in the dark at 4°C to preserve the FITC signal and examined promptly, as FITC fluorescence can fade over time.

• Optimization recommendations: The specific conditions for optimal reactivity should be determined by each researcher for their particular tissue and experimental context .

How can researchers address common technical challenges when using AKT3 FITC antibodies?

When working with AKT3 FITC antibodies, researchers may encounter several challenges. Here are evidence-based solutions:

What emerging applications utilize AKT3 FITC antibodies in neurodegenerative disease research?

Recent advancements in neurodegeneration research have opened new applications for AKT3 FITC antibodies:

• Single-cell analysis of neuroinflammation: AKT3 FITC antibodies enable high-resolution analysis of AKT3 expression in specific cell populations during neuroinflammatory processes. This is particularly relevant given the evidence that Akt3−/− mice have significantly more CD45+ and Iba1+ cells in the spinal cord during EAE .

• Biomarker development: Flow cytometric detection of AKT3 in peripheral blood mononuclear cells may serve as a potential biomarker for neuroinflammatory disease progression, based on the proven role of Akt3 in T-cell function during EAE .

• Therapeutic target validation: As AKT3 has been implicated in neuroprotection during inflammatory demyelination, FITC-conjugated antibodies can help validate the engagement of therapeutic compounds targeting this pathway.

• Neuronal-immune cell interaction studies: By combining AKT3 FITC antibodies with markers for different cell types, researchers can investigate the role of AKT3 in neuronal-immune cell communications during disease.

• Regulatory T-cell therapy monitoring: Given the connection between Akt3 and FOXP3+ regulatory T-cells, AKT3 FITC antibodies could help monitor therapeutic approaches involving Treg augmentation in neuroinflammatory conditions .

The demonstrated upregulation of proinflammatory cytokines IL-2, IL-17, and IFN-γ in Akt3−/− mice relative to wild-type during EAE suggests that monitoring AKT3 expression may provide insights into inflammatory status in these conditions .

How do different experimental models affect the interpretation of AKT3 FITC antibody results?

Different experimental models can significantly impact how AKT3 FITC antibody results should be interpreted:

• Knockout models: Complete Akt3−/− mice show worse EAE outcomes with severe demyelination and increased inflammation, providing a negative control for antibody specificity and a model for studying consequences of AKT3 deficiency .

• Enhanced activity models: Akt3Nmf350 mice with enhanced kinase activity demonstrate protective effects during EAE, offering a complementary model to understand gain-of-function scenarios .

• Conditional knockout models: Cell-specific deletion models such as Akt3 deletion in CD4+ T-cells (showing earlier EAE onset) versus neuronal deletion (showing no EAE outcome difference) help dissect tissue-specific roles of AKT3 .

• Bone marrow chimera models: Wild-type mice receiving Akt3−/− bone marrow show higher clinical scores during EAE than controls receiving wild-type bone marrow, highlighting the importance of AKT3 in peripheral immune cells .

• Cell culture systems: When interpreting AKT3 FITC antibody results in cell lines, consideration should be given to potential differences in expression levels and signaling pathways compared to primary cells.

• Human patient samples: Caution should be exercised when extending findings from mouse models to human samples, though antibodies like K17-H have been validated in human chronic lymphocytic leukemia samples .

Each of these models provides different contexts for interpreting AKT3 detection, and researchers should select appropriate controls based on their specific experimental system.

What novel research questions about AKT3 function could be addressed using FITC-conjugated antibodies?

FITC-conjugated AKT3 antibodies can facilitate investigation of several emerging research questions:

• Isoform-specific signaling dynamics: How do activation kinetics and subcellular localization of AKT3 differ from other AKT isoforms in response to various stimuli, particularly in neural tissues?

• Role in synaptic plasticity: Given AKT3's importance in neural development and cognitive functions , how does its expression and phosphorylation state change during learning and memory formation?

• Immunometabolic regulation: How does AKT3 influence metabolic programming in different T-cell subsets during neuroinflammation, and how does this differ from other AKT isoforms?

• Blood-brain barrier interaction: Does AKT3 expression in immune cells affect their ability to cross the blood-brain barrier during neuroinflammatory conditions?

• Therapeutic target identification: Can selective modulation of AKT3 in T-cells provide neuroprotection without affecting essential functions of other AKT isoforms in different tissues?

• Biomarker development: Could flow cytometric analysis of AKT3 expression patterns in peripheral immune cells serve as a biomarker for neuroinflammatory disease progression or treatment response?

The accumulating evidence implicating AKT3 in neurodegenerative disorders, psychiatric conditions, and certain cancers suggests that addressing these questions could have significant translational impact.

How might advances in flow cytometry technology enhance AKT3 detection and analysis?

Emerging flow cytometry technologies offer new opportunities for AKT3 analysis:

• Spectral flow cytometry: Newer instruments capable of spectral unmixing could permit more complex panels combining AKT3 FITC antibodies with additional markers, enabling deeper phenotyping of cell populations expressing AKT3.

• Mass cytometry (CyTOF): While not utilizing fluorescence, metal-tagged AKT3 antibodies could be incorporated into high-dimensional panels to simultaneously assess dozens of additional markers alongside AKT3.

• Imaging flow cytometry: Combining flow cytometry with microscopy allows assessment of AKT3 subcellular localization patterns alongside quantitative expression data at the single-cell level.

• Phospho-flow cytometry: Integration of AKT3 detection with simultaneous measurement of multiple phosphorylation sites in signaling pathways could provide dynamic information about AKT3 activation states.

• Single-cell sorting with transcriptomics: FITC-based sorting of AKT3-positive cells followed by single-cell RNA sequencing could reveal transcriptional networks associated with different AKT3 expression levels.

The continuously evolving landscape of cytometric technologies promises to enhance our understanding of AKT3 biology through increasingly sophisticated detection and analysis methods.