Phospho-AKT1/AKT2/AKT3 (Y315) Antibody

What is Phospho-AKT1/AKT2/AKT3 (Y315) Antibody and what epitope does it recognize?

Phospho-AKT1/AKT2/AKT3 (Y315) Antibody is a rabbit polyclonal antibody that specifically recognizes AKT proteins when phosphorylated at tyrosine residue 315 (Y315) in AKT1, or the equivalent residues in AKT2 (Y316) and AKT3 (Y312). This antibody is generated using a synthesized phosphopeptide derived from human AKT around the phosphorylation site of Y315 as the immunogen . It is affinity-purified from rabbit antiserum by affinity-chromatography using the epitope-specific immunogen, enhancing its specificity for the phosphorylated form of this site .

What is the significance of Y315 phosphorylation in AKT signaling pathways?

While the phosphorylation of AKT at serine 473 (S473) and threonine 308 (T308) are well-characterized and known to be essential for full AKT activation, the tyrosine 315 (Y315) phosphorylation represents an additional regulatory mechanism in AKT signaling. Y315 phosphorylation likely contributes to the fine-tuning of AKT activity and may play roles in specific cellular contexts or in response to particular stimuli . Understanding Y315 phosphorylation provides insights into the nuanced regulation of AKT signaling beyond the canonical activation sites, potentially revealing new therapeutic targets or biomarkers for diseases involving dysregulated AKT signaling .

How does this antibody differ from those targeting other AKT phosphorylation sites?

The Phospho-AKT1/AKT2/AKT3 (Y315) Antibody specifically recognizes the tyrosine 315 phosphorylation site, distinguishing it from more commonly used antibodies that target the S473 or T308 phosphorylation sites. Each phospho-specific antibody provides information about a different aspect of AKT regulation:

• Antibodies targeting T308 detect AKT phosphorylated at threonine 308, which is mediated by PDK1 and is necessary for partial activation

• Antibodies targeting S473 detect AKT phosphorylated at serine 473, which is mediated by mTORC2 and is required for full activation

• The Y315 antibody detects AKT phosphorylated at tyrosine 315, providing information about additional regulatory mechanisms

Using these antibodies in combination can provide a more comprehensive picture of AKT activation status in different experimental contexts or disease states .

What research applications has this antibody been validated for?

The Phospho-AKT1/AKT2/AKT3 (Y315) Antibody has been validated for several research applications, including:

• Western blot (WB): For detecting phosphorylated AKT proteins in cell or tissue lysates with recommended dilutions of 1:500-1:1000

• Immunohistochemistry (IHC): For visualizing phosphorylated AKT in fixed tissue sections with recommended dilutions of 1:100-1:300

• Immunocytochemistry/Immunofluorescence (ICC/IF): For analyzing phosphorylated AKT localization in fixed cells with recommended dilutions of 1:100-1:500

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of phosphorylated AKT with recommended dilutions of 1:20000

Each application provides different types of information about AKT phosphorylation and may require specific optimization for particular experimental systems .

How should I optimize Western blot protocols using this antibody?

To optimize Western blot protocols using the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody, consider the following strategies:

• Sample preparation: Preserve phosphorylation by using phosphatase inhibitors in your lysis buffer and keep samples cold throughout processing.

• Loading controls: Include appropriate loading controls such as total AKT or housekeeping proteins to normalize phospho-AKT signals.

• Membrane blocking: Use 3-5% BSA in TBS-T rather than milk, as milk contains phosphatases that can dephosphorylate your targets.

• Antibody dilution: Start with the recommended dilution (1:500-1:1000) and adjust based on signal intensity and background .

• Positive controls: Include a positive control, such as cell lysates from cells treated with growth factors known to induce AKT phosphorylation (e.g., EGF treatment of HepG2 cells has been shown to increase phosphorylation) .

• Detection system: Use a sensitive detection system appropriate for your experimental needs, such as chemiluminescence for general detection or fluorescence-based methods for more precise quantification.

• Validation steps: Confirm specificity using phospho-blocking peptides or phosphatase treatment of control samples to verify that the signal is truly phospho-specific .

What controls should I include when using this antibody in immunohistochemistry or immunofluorescence?

When using the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody for immunohistochemistry or immunofluorescence, include these essential controls:

• Positive tissue control: Include a tissue sample known to express phosphorylated AKT at Y315, such as breast carcinoma tissue as demonstrated in published results .

• Blocking peptide control: Perform a parallel staining where the antibody is pre-incubated with the phospho-peptide used as the immunogen. This should abolish specific staining, as demonstrated in immunohistochemical analysis of human breast carcinoma tissue .

• Negative control: Include a tissue sample where phosphorylated AKT at Y315 is expected to be minimal or absent, or omit the primary antibody but include all other steps to assess background from the secondary detection system.

• Phosphatase treatment control: Treat a section with phosphatase before staining to confirm phospho-specificity of the signal.

• Methanol fixation considerations: For immunofluorescence in cell lines such as HeLa cells, methanol fixation has been successfully used with this antibody at a 1:100 dilution .

• Antigen retrieval optimization: For paraffin-embedded tissues, heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) has been shown to be effective .

Does this antibody distinguish between the three AKT isoforms?

The antibody was designed to target a conserved region surrounding the phosphorylation site across all three isoforms, resulting in its ability to recognize AKT1, AKT2, and AKT3 when appropriately phosphorylated . If isoform-specific information is needed, additional techniques would be required, such as immunoprecipitation with isoform-specific antibodies followed by detection with this phospho-specific antibody, or the use of cells with selective knockdown of specific AKT isoforms.

What is the species cross-reactivity of this antibody?

According to the search results, the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody has confirmed reactivity with samples from the following species:

• Human

• Mouse

• Rat

Some sources indicate broader reactivity that may include additional species, though these would require validation by the researcher. The cross-reactivity is due to the high conservation of the sequence surrounding the Y315 phosphorylation site across these species .

When working with species not explicitly listed as reactive, preliminary validation experiments are advisable to confirm antibody performance, particularly if the sequence around the phosphorylation site is known to be conserved in that species.

How can I validate the specificity of this antibody in my experimental system?

To validate the specificity of the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody in your experimental system, consider these approaches:

• Phosphatase treatment: Treat one set of samples with a phosphatase enzyme before immunoblotting or staining. The signal should disappear if it is truly phospho-specific .

• Blocking peptide competition: Pre-incubate the antibody with the phospho-peptide used as the immunogen. This should abolish specific binding, as demonstrated in immunohistochemical analysis results .

• Pharmacological inhibition: Treat cells with PI3K/AKT pathway inhibitors and verify that the signal decreases, or stimulate with activators (like EGF as shown in HepG2 cell experiments) and verify that the signal increases .

• Comparative analysis: Compare the pattern of Y315 phosphorylation with other well-characterized AKT phosphorylation sites (S473, T308) under various conditions to ensure the observed changes make biological sense .

• Western blot band verification: Confirm that the detected band appears at the expected molecular weight (approximately 56-60 kDa, with the observed band size reported as 60 kDa in U87MG cell lysates) .

• Controls in staining applications: In immunofluorescence or immunohistochemistry, include positive controls (like A549 or HeLa cells for immunofluorescence, or breast carcinoma tissue for IHC) where the phosphorylation has been previously demonstrated .

How can I use this antibody to investigate AKT signaling in cancer models?

The Phospho-AKT1/AKT2/AKT3 (Y315) Antibody can be valuable for investigating AKT signaling in cancer models through several approaches:

• Cancer type profiling: Use the antibody to analyze Y315 phosphorylation across different cancer cell lines or patient-derived samples. The antibody has been successfully used with cancer cell lines including HepG2, U87MG (glioma), and A549 (lung cancer), as well as in breast carcinoma tissue .

• Drug response studies: Monitor changes in Y315 phosphorylation following treatment with therapeutic agents targeting the PI3K/AKT pathway. The assay's high sensitivity allows measurement of activated AKT from very small samples, making it suitable for monitoring patient response to targeted therapies .

• Pathway analysis: Investigate the relationship between Y315 phosphorylation and other signaling events in cancer cells, particularly in the context of the PI3K/AKT pathway which is frequently dysregulated in multiple cancers, including breast cancer .

• Correlation with malignant phenotypes: Assess whether Y315 phosphorylation correlates with cancer cell behaviors such as proliferation, survival, or invasion. This is particularly relevant for AKT3, which has been reported to be crucial for the viability of malignant glioma cells .

• Spatial distribution analysis: In tissue sections, analyze the localization of Y315 phosphorylation relative to tumor architecture using immunohistochemistry, which has been validated for this antibody .

The ability to detect and measure AKT phosphorylation from very small amounts of protein allows for accurate evaluation of patient response to drugs targeting activated PI3K-AKT using limited clinical specimens .

What approaches can I use to quantify changes in Y315 phosphorylation relative to total AKT levels?

To accurately quantify changes in Y315 phosphorylation relative to total AKT levels, consider these methodological approaches:

• Western blot with ratio analysis: Probe parallel blots or strip and reprobe the same blot with the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody and a total AKT antibody. Use densitometry to calculate the ratio of phosphorylated to total AKT, normalizing for any changes in total AKT expression .

• ELISA-based approaches: The antibody has been validated for ELISA applications (1:20000 dilution), allowing for quantitative determination of phosphorylated AKT levels . Paired with a total AKT ELISA, this can provide precise phosphorylation ratios.

• Immunofluorescence quantification: For cellular analyses, perform quantitative immunofluorescence with the phospho-AKT antibody, counterstain for total AKT, and use image analysis software to determine the phospho-to-total ratio on a per-cell basis .

• Specialized protein separation techniques: Advanced techniques mentioned in the search results can separate protein molecules based on their isoelectric point, allowing for detection and quantitation of AKT isoforms and phosphoforms in samples from as few as 56 cells .

• Western blot validation: When performing quantitative Western blot analysis, verify that you observe the expected band size (the predicted band size is 56 kDa, though an observed band size of 60 kDa has been reported in some cell lines) .

These approaches allow for normalization of phosphorylation changes to total protein levels, providing more meaningful data about the activation state of the AKT pathway rather than just absolute phosphorylation levels.

How does Y315 phosphorylation relate to other phosphorylation sites on AKT?

The relationship between Y315 phosphorylation and the canonical AKT phosphorylation sites (S473 and T308) is an area that requires further investigation, but we can draw some insights from the available information:

• Activation sequence: While T308 phosphorylation has been shown to partially activate AKT, and additional phosphorylation on S473 is necessary for full activation , the precise role of Y315 phosphorylation in this activation sequence is less well-characterized.

• Comparative analysis possibilities: The availability of antibodies specific for different phosphorylation sites allows researchers to compare Y315 phosphorylation patterns with those of S473 and T308 under various conditions. For example, the search results mention that peaks 5.46, 5.54, and 5.58 were detected by a pS473 antibody in one analysis system .

• Potential for distinct regulation: Y315, being a tyrosine residue rather than serine or threonine, is likely phosphorylated by different kinases than those responsible for S473 and T308 phosphorylation. This suggests potentially distinct regulatory mechanisms and responses to stimuli.

• Functional implications: AKT3 (one of the isoforms recognized by this antibody) plays an important role in brain development and is crucial for the viability of malignant glioma cells . Understanding how Y315 phosphorylation contributes to these functions, potentially in coordination with other phosphorylation events, represents an important research direction.

• Methodological considerations: When designing experiments to study the relationship between different phosphorylation sites, researchers should consider using multiple antibodies in parallel analyses and correlating the patterns with functional outcomes to build a more complete picture of AKT regulation.

What positive and negative controls should I include when studying AKT Y315 phosphorylation?

When studying AKT Y315 phosphorylation, including appropriate positive and negative controls is essential for experimental rigor:

Positive controls:

• Growth factor stimulation: Treat cells with growth factors known to activate the PI3K/AKT pathway. The search results specifically mention EGF treatment of HepG2 cells as a positive control that increases phosphorylation at this site .

• Known positive samples: Include cell lines or tissue samples known to exhibit high levels of AKT Y315 phosphorylation. The search results mention several cell lines where this phosphorylation has been detected, including HepG2, U87MG, A549, and HeLa cells .

• Cancer tissue samples: Certain cancer tissues, such as breast carcinoma, have been demonstrated to show positive staining for this phosphorylation site in immunohistochemical analyses .

Negative controls:

• Blocking peptide control: Pre-incubate the antibody with the phospho-peptide used as the immunogen. This should abolish specific staining and can be used to confirm signal specificity, as demonstrated in immunohistochemical analysis of human breast carcinoma tissue .

• Phosphatase treatment: Treat a portion of your sample with phosphatase to dephosphorylate proteins, which should eliminate the phospho-specific signal.

• Pathway inhibition: Treat cells with PI3K/AKT pathway inhibitors to suppress AKT phosphorylation.

Technical controls:

• Secondary antibody only: Omit the primary antibody to assess non-specific binding of the secondary antibody or detection system.

• Dilution series: Test a range of antibody dilutions to identify the optimal concentration that maximizes specific signal while minimizing background.

• Multiple detection methods: When possible, confirm key findings using different techniques (e.g., both Western blot and immunofluorescence) to rule out method-specific artifacts.

How should I interpret contradictory results between different phosphorylation sites on AKT?

When encountering contradictory results between different phosphorylation sites on AKT (e.g., Y315 vs. S473 or T308), consider these interpretative frameworks:

• Site-specific regulation: Different phosphorylation sites may be regulated by distinct upstream kinases or phosphatases. While S473 phosphorylation is mediated by mTORC2 and T308 by PDK1 , Y315 phosphorylation likely involves tyrosine kinases, potentially leading to different responses to the same stimulus.

• Temporal dynamics: The sites may be phosphorylated or dephosphorylated with different kinetics. For example, one phosphorylation event might occur rapidly following stimulation while another might be delayed or more sustained.

• Spatial regulation: Phosphorylation events may occur in different subcellular compartments. Immunofluorescence studies with the Y315 antibody in cell lines like HeLa can provide valuable information about the spatial distribution of this phosphorylation event relative to other AKT modifications .

• Isoform specificity: The antibody recognizes all three AKT isoforms (AKT1, AKT2, and AKT3) , but these isoforms may be differentially regulated. AKT3, for instance, plays specific roles in brain development and glioma cell viability that may not be shared by the other isoforms .

• Functional context: Consider how the contradictory phosphorylation patterns relate to downstream pathway activation. For example, while S473 phosphorylation is widely used to assess AKT activity and is necessary for full activation , the specific role of Y315 phosphorylation in modulating AKT function may be different.

• Technical considerations: Verify that each phospho-specific antibody is working properly with appropriate controls, as described in previous sections. The Phospho-AKT1/AKT2/AKT3 (Y315) Antibody has been validated in multiple applications and cell types , but each experimental system may require specific optimization.

Rather than viewing contradictory results as problematic, consider them as potential insights into the complex regulation of AKT in your specific biological context.

What are common pitfalls in interpreting data from phospho-specific antibodies?

Interpreting data from phospho-specific antibodies like the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody comes with several potential pitfalls:

• Antibody specificity issues: Always validate that the observed signal is truly phospho-specific using controls such as blocking peptides or phosphatase treatment. The search results demonstrate the use of a blocking peptide in IHC applications that abolished the signal, confirming specificity .

• Sample preparation effects: Phosphorylation status can be rapidly altered during sample preparation. Use phosphatase inhibitors and cold processing to preserve phosphorylation states. The antibody is typically provided in a buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, and should be stored at -20°C or -80°C with repeated freeze-thaw cycles avoided .

• Quantification challenges: When quantifying Western blot signals, ensure proper normalization to total protein or loading controls. Observed band size may differ slightly from predicted size (predicted: 56 kDa; observed: 60 kDa in some cell lines) .

• Biological complexity misinterpretation: Changes in phosphorylation may not always directly correlate with functional outcomes. AKT signaling involves multiple isoforms (AKT1, AKT2, AKT3) that regulate many processes including metabolism, proliferation, cell survival, growth, and angiogenesis , so phosphorylation at a single site provides only partial information about pathway activity.

• Context neglect: Interpretation without considering cellular context can be misleading. For example, AKT3 (recognized by this antibody) plays a specific role in brain development and glioma cell viability that may not be relevant in all tissues.

• Technical limitations: Different detection methods have different sensitivities. The antibody has been validated for multiple applications (WB, IHC, ICC/IF, ELISA) with specific recommended dilutions for each, but these may need optimization for your specific experimental system.

• Correlation vs. causation confusion: Changes in Y315 phosphorylation correlating with a phenotype don't necessarily prove a causal relationship. Additional experiments, such as mutation of the phosphorylation site or specific inhibition of the responsible kinase, would be needed to establish causality.

By being aware of these potential pitfalls and implementing appropriate controls and validation steps, researchers can generate more reliable and biologically meaningful data using phospho-specific antibodies.

What are the optimal storage conditions for this antibody?

For optimal performance and longevity of the Phospho-AKT1/AKT2/AKT3 (Y315) Antibody, follow these storage guidelines:

• Storage temperature: Store the antibody at -20°C or -80°C upon receipt . This low temperature helps preserve the antibody's activity and specificity.

• Formulation: The antibody is typically provided in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide , which helps maintain stability during storage.

• Avoid freeze-thaw cycles: Repeated freezing and thawing can degrade the antibody and reduce its performance. The product information specifically notes "Avoid repeated freeze" .

• Aliquoting recommendation: If frequent use is anticipated, prepare small aliquots upon receipt and store them separately to minimize the number of freeze-thaw cycles each portion undergoes.

• Handling during use: When retrieving from storage, thaw the antibody on ice and return any unused portion to the freezer promptly. Keep the antibody cold during experimental procedures.

• Working dilution storage: If preparing working dilutions, they can typically be stored at 4°C for short periods (1-2 weeks), but for longer storage, keeping aliquots at -20°C or -80°C is recommended.

• Transport considerations: If the antibody needs to be transported between laboratories, use dry ice to maintain the cold chain and prevent degradation.