AKT1 (Ab-129) Antibody

What is AKT1 (Ab-129) Antibody and what epitope does it recognize?

AKT1 (Ab-129) Antibody is a rabbit polyclonal antibody that specifically recognizes AKT1 around the phosphorylation site of serine 129 (S129). The immunogen used for its development is a synthesized non-phosphopeptide derived from human Akt with the amino acid sequence surrounding the S129 phosphorylation site (D-N-S(p)-G-A) . This antibody is designed to detect both phosphorylated and non-phosphorylated forms of AKT1 at this specific region, making it valuable for studying AKT1 regulation through S129 phosphorylation.

What experimental applications has AKT1 (Ab-129) Antibody been validated for?

AKT1 (Ab-129) Antibody has been validated for multiple experimental applications:

• Western Blot (WB): Recommended dilution range of 1:500-1:3000

• Immunohistochemistry (IHC-P): Recommended dilution range of 1:50-1:100

• Immunofluorescence (IF): Recommended dilution range of 1:100-1:500

• Enzyme-Linked Immunosorbent Assay (ELISA)

The antibody has demonstrated successful detection of AKT1 in various experimental systems, including Western blot analysis of HuvEc cells and JK cells, immunohistochemical staining of human skeletal muscle tissue, and immunofluorescence analysis of HeLa cells .

What species reactivity has been confirmed for this antibody?

AKT1 (Ab-129) Antibody has been experimentally confirmed to react with:

• Human samples

• Mouse samples

• Rat samples

This broad species reactivity makes it versatile for comparative studies across different model organisms.

What are the optimal storage conditions for maintaining AKT1 (Ab-129) Antibody activity?

For optimal antibody performance and stability:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Formulation: The antibody is supplied in rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol

Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity. For experiments requiring frequent use, it is recommended to keep a working aliquot at 4°C for daily experimentations while storing the remaining antibody at -20°C or -80°C .

How should I optimize Western blot conditions when using AKT1 (Ab-129) Antibody?

For optimal Western blot results with AKT1 (Ab-129) Antibody:

• Sample preparation:

  • Use lysis buffer containing 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 3 mM EGTA, 5 mM EDTA

  • Include phosphatase inhibitors (10 mM sodium pyrophosphate, 5 mM sodium orthovanadate, 5 mM sodium fluoride, 10 μM okadaic acid)

  • Add protease inhibitor mixture and 1 mM phenylmethylsulfonyl fluoride

• Antibody dilution:

  • Start with 1:1000 dilution and adjust based on signal strength

  • Titrate between 1:500-1:3000 for optimal signal-to-noise ratio

• Expected molecular weight:

  • Look for a band at approximately 60-65 kDa

• Positive controls:

  • HuvEc cells and JK cells have shown good reactivity with this antibody

  • For phospho-specific detection, PMA-treated A549 lysates may be used as positive controls

What experimental controls should be included when using this antibody for phosphorylation studies?

When studying AKT1 phosphorylation at S129, include these essential controls:

• Phospho-peptide competition: Incubate the antibody with the phosphorylated peptide antigen prior to application to confirm specificity for the phosphorylated form.

• Non-phosphorylated peptide control: Include a control using the non-phosphorylated peptide to demonstrate phospho-specificity, as shown in validation experiments with AKT1-S129 antibodies .

• Phosphatase treatment control: Treat part of your sample with lambda phosphatase to demonstrate that the signal depends on phosphorylation status.

• Kinase activation/inhibition: Use CK2 activators or inhibitors to manipulate the phosphorylation status of AKT1 at S129, as CK2 is known to phosphorylate this site .

• Knockout/knockdown samples: Where available, include AKT1 knockout or knockdown samples as negative controls to confirm antibody specificity .

How does phosphorylation at S129 affect AKT1 function and what pathways are implicated?

AKT1 phosphorylation at S129 has several documented functional implications:

• Kinase activity enhancement: Phosphorylation of AKT1 at Ser129 by protein kinase CK2 promotes association of AKT1 with the HSP90 chaperone. This interaction enhances AKT1 kinase activity by inhibiting dephosphorylation of AKT1 at Thr308 .

• Signaling pathway regulation: S129 phosphorylation can enhance β-catenin transcriptional activity, potentially affecting Wnt signaling pathway outputs .

• Cell survival: CK2-mediated phosphorylation of AKT1 at S129 has been linked to enhanced cancer cell survival mechanisms .

• Stability regulation: This phosphorylation may contribute to AKT1 protein stability and influence its half-life in cells.

• Integrin activation: Akt1 signaling has been implicated in inside-out activation of integrins in endothelial cells and fibroblasts, which mediates matrix assembly and recognition, though the specific role of S129 phosphorylation in this process requires further investigation .

How can I differentiate between AKT1, AKT2, and AKT3 isoforms in my experiments?

Differentiating between AKT isoforms requires careful experimental design:

• Isoform-specific antibodies: Use antibodies that specifically recognize unique epitopes in each AKT isoform. AKT1 (Ab-129) targets a region specific to AKT1.

• Knockout/knockdown validation: Utilize cells derived from isoform-specific knockout mice (e.g., Akt1−/−) or cells with siRNA-mediated knockdown of specific isoforms to validate antibody specificity .

• Molecular weight differences: Though subtle, the different AKT isoforms may migrate slightly differently on SDS-PAGE (AKT1: ~60 kDa, AKT2: ~56 kDa, AKT3: ~62 kDa).

• Functional assays: As demonstrated in studies of experimental autoimmune encephalomyelitis, AKT1 and AKT2 can have opposing functions. Akt1−/− mice develop ameliorated EAE, whereas Akt2−/− mice develop exacerbated EAE compared to wild-type mice .

• Tissue expression patterns: Consider the differential expression patterns of AKT isoforms across tissues when designing experiments.

What is the relationship between AKT1 S129 phosphorylation and T308/S473 phosphorylation sites?

The interrelationship between AKT1 phosphorylation sites reveals complex regulatory mechanisms:

• Hierarchical regulation: While T308 (PDK1-mediated) and S473 (mTORC2-mediated) phosphorylation are primary activating events for AKT1, S129 phosphorylation by CK2 serves as a secondary regulatory mechanism.

• Stabilization effect: S129 phosphorylation promotes association with HSP90 chaperone, which specifically inhibits dephosphorylation of AKT1 at T308, thus maintaining AKT1 in an active state for longer periods .

• Independent regulation: Unlike T308 and S473 phosphorylation, which are regulated by growth factor signaling through PI3K activation, S129 phosphorylation by CK2 can occur independently of PI3K activity.

• Functional synergy: The full activation of AKT1 typically requires phosphorylation at multiple sites, with S129 phosphorylation potentially enhancing the effects of T308/S473 phosphorylation.

• Technical considerations: When studying AKT1 activation, researchers should consider monitoring multiple phosphorylation sites simultaneously to gain comprehensive insights into AKT1 activation status.

What are the common challenges in detecting AKT1 S129 phosphorylation and how can they be addressed?

Researchers frequently encounter these challenges when detecting AKT1 S129 phosphorylation:

• Low basal phosphorylation levels: S129 phosphorylation may be present at low levels under basal conditions.

  • Solution: Use stimuli known to enhance CK2 activity or S129 phosphorylation, such as PMA treatment in A549 cells .

• Rapid dephosphorylation during sample preparation:

  • Solution: Include comprehensive phosphatase inhibitor cocktails in lysis buffers (sodium pyrophosphate, sodium orthovanadate, sodium fluoride, and okadaic acid) .

• Cross-reactivity with other phosphorylated proteins:

  • Solution: Validate antibody specificity using peptide competition assays and AKT1 knockout/knockdown samples .

• Signal masking by abundant non-phosphorylated protein:

  • Solution: Consider enrichment of phosphorylated proteins using phospho-specific immunoprecipitation prior to Western blotting.

• Variability in phosphorylation levels across cell types:

  • Solution: Establish baseline phosphorylation levels for your specific cell type and optimize detection conditions accordingly.

How can I use AKT1 (Ab-129) Antibody in immunofluorescence studies to visualize AKT1 subcellular localization?

For successful immunofluorescence studies with AKT1 (Ab-129) Antibody:

• Cell preparation:

  • Culture cells on coverslips or chamber slides

  • Fix with 4% paraformaldehyde or methanol depending on epitope sensitivity

  • Permeabilize with 0.1-0.5% Triton X-100 for intracellular epitope access

• Antibody application:

  • Use a dilution range of 1:100-1:500

  • Incubate overnight at 4°C or for 1-2 hours at room temperature

  • HeLa cells have been successfully used for IF with this antibody

• Visualization strategies:

  • Use fluorophore-conjugated secondary antibodies specific to rabbit IgG

  • Include counterstains for nuclei (DAPI) and potentially other cellular structures

  • Consider co-staining with markers for specific cellular compartments (e.g., plasma membrane, endosomes, nucleus) to assess AKT1 localization

• Controls:

  • Include secondary-only controls to assess background

  • Consider siRNA knockdown of AKT1 as a negative control

  • Use stimuli known to alter AKT1 localization (e.g., growth factor stimulation) as positive controls

How do I design experiments to study the functional significance of AKT1 S129 phosphorylation?

To investigate the functional significance of AKT1 S129 phosphorylation:

• Site-directed mutagenesis approaches:

  • Generate S129A (phospho-deficient) and S129D/E (phospho-mimetic) AKT1 mutants

  • Express these mutants in AKT1 knockout or knockdown backgrounds

  • Assess effects on AKT1 kinase activity, protein stability, and downstream signaling

• Pharmacological manipulation:

  • Utilize CK2 inhibitors to decrease S129 phosphorylation

  • Monitor effects on AKT1 downstream targets and cellular functions

• Cellular function assays based on known AKT1 roles:

  • Cell migration/wound healing assays (AKT1 has been implicated in cell motility)

  • Integrin activation assays using WOW-1 Fab or HUTS-4 binding

  • Matrix assembly assays, as AKT1 regulates integrin activation and matrix recognition

  • Cell survival assays under stress conditions

• In vivo models:

  • Consider utilizing Akt1−/− mice and rescue experiments with wild-type or mutant (S129A or S129D/E) AKT1

  • Examine phenotypes relevant to AKT1 function, such as susceptibility to autoimmune conditions like experimental autoimmune encephalomyelitis

• Correlation with disease states:

  • Analyze S129 phosphorylation status in disease models or patient samples

  • Correlate findings with disease progression or therapeutic responses

What is the current understanding of differential roles between AKT isoforms in disease models?

Recent research has revealed distinct and sometimes opposing functions of AKT isoforms:

• Autoimmune diseases: In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis:

  • Akt1−/− mice develop ameliorated EAE

  • Akt2−/− mice develop exacerbated EAE compared to wild-type mice

  • These differential effects appear to be mediated through regulation of thymus-derived regulatory T cell (tTreg) proliferation

  • Akt-1 inhibits tTreg proliferation, facilitating antigen-specific Th1/Th17 responses

  • Akt-2 potentiates tTreg proliferation and suppresses antigen-specific Th1/Th17 responses

• Cancer contexts:

  • AKT1 has been implicated in promoting certain aspects of tumorigenesis

  • S129 phosphorylation specifically enhances β-catenin transcriptional activity and cancer cell survival

• Vascular biology:

  • Akt1 is essential for inside-out activation of integrins in endothelial cells and fibroblasts

  • This activation mediates matrix assembly and recognition, which has implications for vascular development and function

• Therapeutic implications:

  • Targeting Akt-1 specifically has shown promise as a potential therapeutic approach for multiple sclerosis

  • Isoform-specific inhibition may provide more precise therapeutic strategies with fewer side effects than pan-AKT inhibition

How can phospho-specific antibodies like AKT1 (Ab-129) be integrated into phosphoproteomics workflows?

Integrating phospho-specific antibodies into phosphoproteomics workflows offers several advantages:

• Targeted phosphopeptide enrichment:

  • Use AKT1 (Ab-129) Antibody for immunoprecipitation prior to mass spectrometry analysis

  • Enhance detection sensitivity for low-abundance AKT1 phospho-forms

• Validation of mass spectrometry findings:

  • Confirm phosphoproteomics hits using Western blotting with phospho-specific antibodies

  • Quantify relative abundance of specific phosphorylation events across multiple samples

• Spatial phosphoproteomics:

  • Combine immunofluorescence using AKT1 (Ab-129) Antibody with subcellular fractionation approaches

  • Map compartment-specific phosphorylation patterns of AKT1

• Temporal dynamics studies:

  • Use phospho-specific antibodies to track rapid phosphorylation changes in time-course experiments

  • Complement mass spectrometry data with higher temporal resolution Western blot analysis

• Single-cell phosphorylation analysis:

  • Apply phospho-specific antibodies in flow cytometry or imaging cytometry approaches

  • Assess cell-to-cell variability in AKT1 phosphorylation states within heterogeneous populations

What emerging techniques could enhance the utility of AKT1 (Ab-129) Antibody in future research?

Several cutting-edge techniques could expand the research applications of AKT1 (Ab-129) Antibody:

• Proximity labeling approaches:

  • Combine with BioID or APEX2 proximity labeling to identify proteins interacting specifically with phosphorylated AKT1 at S129

  • Map phosphorylation-dependent interactomes

• Single-molecule imaging:

  • Use fluorescently labeled AKT1 (Ab-129) Antibody fragments for super-resolution microscopy

  • Track the dynamics of individual phosphorylated AKT1 molecules in living cells

• Intrabodies and nanobodies:

  • Develop cell-permeable antibody derivatives to track AKT1 phosphorylation in living cells

  • Engineer genetically encoded sensors based on AKT1 (Ab-129) binding properties

• Spatial transcriptomics correlation:

  • Integrate immunofluorescence data using AKT1 (Ab-129) Antibody with spatial transcriptomics

  • Correlate AKT1 phosphorylation patterns with gene expression profiles in tissue sections

• Multiplexed antibody-based imaging:

  • Incorporate AKT1 (Ab-129) Antibody into multiplexed immunofluorescence panels

  • Simultaneously detect multiple phosphorylation sites and downstream targets to build comprehensive signaling maps