Kinase Akt Assay Overview

Kinase signaling plays a pivotal role in coupling external stimuli to changes in downstream physiological functions such as metabolism, proliferation and survival in healthy cells. Dysregulation of signal transduction pathways is associated with cancer initiation, progression and recurrence. Akt is a serine/threonine protein kinase that is upregulated across various cancer types and has been extensively investigated as a target for oncology therapeutics due to its mechanistic association with aberrant cell growth, apoptosis and metabolism. 

Inhibitors of the Akt signaling network have shown clinical promise as monotherapies, or in combination with conventional chemotherapeutic drugs to improve efficacy in resistant cancers. Identifying these inhibitors can be challenging due to limitations in standard approaches to measuring kinase activity. Studying dynamic changes in kinase activity can be particularly difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking.  

Traditional methods of studying kinase activity are challenging, due to:

  • Tedious, manual sample preparation
  • End-point assays limiting measurements to a single time point
  • Inability to monitor effects of multiple treatments within the same cells
  • Limitations on treatments to be studied due to lack of throughput

With the Incucyte® Kinase Akt Assay, researchers can now measure and analyze dynamic changes in Akt activity in live cells in a physiologically relevant context. Gain deeper insight into treatment effects through coupling long-term evaluation of protein kinase activity with effects on cell proliferation.
 


Application

Introducing the Incucyte® Kinase Akt Assay for Live-Cell Analysis

The Incucyte® Kinase Akt Green/Red Lentivirus enables efficient, non-perturbing and homogenous labeling of mammalian cells for in vitro analysis of dynamic changes in Akt kinase activity. The lentivirus reagent is a single-cassette, genetically encoded sensor based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent protein-tagged nuclear marker to denote the nuclear/cytoplasmic boundary.

Upon phosphorylation by Akt, the green fluorescent sensor translocates from the nucleus to the cytoplasm. Inhibition of Akt prevents phosphorylation of the sensor and causes it to be retained in the nucleus. This assay offers a powerful solution for measuring and analyzing dynamic changes in Akt activity in live cells over time in a physiologically relevant environment. 

Key Advantages

  • Novel, non-perturbing reagent – Express a stable, genetically-encoded kinase activity reporter using Incucyte® Kinase Akt Lentivirus in a variety of cell types 
  • Unlock your productivity - Bridge the gap between single, target-focused biochemical assays and lower-throughput cell-based follow-up experiments for the identification of novel treatments
  • Conduct temporal studies of Akt activity - Gain new insights with sensitive detection of dynamic changes in Akt activity in live cells over time 
  • Concurrent cell counting - Quantify Akt kinase activity and proliferation within the same assay

Novel, non-perturbing reagent 

Express a stable, genetically-encoded kinase activity reporter using Incucyte® Kinase Akt Lentivirus in a variety of cell types to monitor Akt kinase activity in live cells over time. 


Figure 1. Measure compound effects on Akt activity in live cells.  SK-OV-3 cells stably expressing the Incucyte® Kinase Akt Green/Red indicator were treated with Akt selective inhibitor MK2206, resulting in translocation of the green fluorescent sensor from the cytoplasm to the nucleus.  The kinetic graph on the left shows a decrease in the Nuclear Translocation Ratio over time, indicating Akt inhibition.  The image panel shows the phase and red fluorescence image channels on the top and the green fluorescence channel on the bottom.  Movement of the green fluorescent sensor from the cytoplasm to the nucleus can be seen over 28 minutes, while localization of the red fluorescent nuclear marker does not change. 

Unlock your productivity  

Enhance productivity by evaluating multiple compounds or treatments in a 96-well format.  Bridge the gap between single target-focused biochemical assays and lower-throughput cell-based follow-up experiments for the identification of novel treatments.   


Figure 2. Use live-cell kinetic data to evaluate time-dependent effects on Akt activity of multiple compounds from a single 96-well plate.  A549 cells stably expressing the Incucyte® Kinase Akt Green/Red indicator were seeded in a 96-well plate at 5K cells/well.  Cells were treated with inhibitors targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103.  The 96-well Microplate Graph (left) shows time- and concentration-dependent decreases in the Nuclear Translocation Ratio (NTR) for all compounds tested, indicating Akt inhibition. Kinetic graphs of the NTR over 24 h for AZD5363 and LY294002 (middle) show varying kinetic profiles.  Addition of AZD5363 caused sustained inhibition of Akt over 24 h, while PI3K inhibitor LY294002 caused a transient decrease in NTR followed by a recovery to baseline, indicating reactivation of Akt.  IC50 curves (right) depict 1.5 h and 24 h time point data. 

Conduct temporal studies of Akt activity

Gain new insights with sensitive detection of both short term and long term dynamic changes in Akt activity in live cells continuously over time. Automatically measure and visualize inhibition of Akt and utilize kinetic measurements from any time point.  


Figure 3. Observe effects of serum starvation and Akt activation over time.  HeLa cells stably expressing the Incucyte® Kinase Akt Green/Red indicator were shifted into media containing no serum to remove various growth factors which are known to activate the PI3K/Akt kinase pathway.  The serum shift caused a decrease in the Nuclear Translocation Ratio (NTR), indicating a decrease in Akt activity, while no change was observed in control wells kept in 10% serum.  After 4 h, cells were treated with either 1.1 ng/mL epidermal growth factor (EGF) or 3.3 nM R3-IGF-1 (a recombinant analog of insulin-like growth factor).  Both compounds induced activation of Akt, as demonstrated by a rapid increase in the NTR.  R3-IGF-1 addition resulted in sustained activation over the 12-hour time course, while activation by EGF diminished over time. Representative images from all conditions are shown.  Prior to compound addition, the sensor is localized to the nucleus of cells in serum free media and the cytoplasm of control cells in 10% serum (3 h).  Upon activation by EGF or R3-IGF-1, the sensor translocates from the nucleus to the cytoplasm (4.5 h).   The sensor returns to the nucleus as Akt activity decreases over time in EGF-treated cells, while it remains in the cytoplasm of R3-IGF-1 treated cells (12 h). 

Concurrent cell counting  

Quantify Akt kinase activity and proliferation within the same assay. Gain insights into the effect of Akt inhibition on cancer cell proliferation, all within the same cell population. 


Figure 4. Make concurrent measurements of Akt activity and cell proliferation within the same assay. MDA-MB-231 and T-47D cells expressing the Incucyte® Kinase Akt Green/Red indicator were seeded in 96-well plates at 2.5K cells/well and 4K cells/well, respectively. Nuclear Translocation Ratio (NTR) as a measure of Akt activity and Red Object Counts as a measure of cell proliferation were monitored concurrently in the same cells following treatment with a selective Akt inhibitor (MK2206), and a protein synthesis inhibitor (Cycloheximide). Kinetic graphs of NTR (top row) demonstrate concentration-dependent inhibition of Akt by MK2206 in both cell lines. Measurements of Red Object Count from the same cells (middle row) reveal differential effects of Akt inhibition by MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition in T-47D cells but little effect in MDA-MB-231 cells. Cycloheximide treatment caused inhibition of cell proliferation without affecting Akt activity in both cell lines.  IC50 graphs of Red Object Count (24 h) and Nuclear Translocation Ratio (72 h) are shown (bottom row).  The IC50 for Akt inhibition as measured by the NTR was similar in both cell types, while the IC50 for Red Object Count is significantly left-shifted in T-47D cells compared to MDA-MB-231 cells.  

Ordering Information

Product

Qty.

Cat. No.

Incucyte® Kinase Akt Green/Red Lentivirus

1 vial (0.2 mL)

BA-04868


Resources

Proliferation

Cell proliferation is the biological process of cells increasing in number over time through cell division ('mitosis' in eukaryotic cells). Proliferat...

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