The Akt signaling pathway, one of the most often dysregulated signaling pathways in cancer, plays an important role in mediating a very broad range of cellular processes such as growth and development, cell cycle, energy homeostasis, and cell survival. Akt, a serine/threonine kinase that phosphorylates over 100 protein substrates, mediating cell survival and proliferation signals, is often itself hyperphosphorylated in various tumor types. Although the Akt gene itself is not known to be mutated in cancers, many of the upstream regulators of Akt, including IR, IRS, PI3K and PTEN, are oncogenes and tumor suppressors. Downstream of Akt, the mammalian Target Of Rapamycin (mTOR) complex is a key regulator of growth and metabolism. As nearly all of the players in the Akt/mTOR signaling pathway are coordinately regulated by phosphorylation, understanding the role of this pathway in normal physiological processes and in diseases such as cancer and diabetes requires the ability to simultaneously measure phosphorylation status of multiple protein targets.
Cross-reactivity between the antibodies and any of the other analytes in this panel is non-detectable or negligible.
Intracellular Bead-Based Multiplex Assays using the Luminex technology enables the simultaneous relative quantitation of multiple phosphorylation and total pathway proteins in tissue and cell lysate samples. Compare Multiplexing results to those of Western blotting.
An overnight (4°C) incubation is recommended for best results.
This assay requires 25 μL diluted cell lysate per well.
This kit must be run using Assay Buffer 2 (provided).
The suggested working range of protein concentration for the assay is 15 to 150 ng of total protein/well (25 μL/well at 600 to 6,000 ng/mL).
- Akt (Ser473)
- GSK3α (Ser21)
- GSK3β (Ser9)
- IGF1R (Tyr1135/Tyr1136)
- IR (Tyr1162/Tyr1163)
- IRS1 (Ser636)
- mTOR (Ser2448)
- p70S6K (Thr412)
- PTEN (Ser380)
- RPS6 (Ser235/Ser236)
- TSC2 (Ser939)
Storage and Stability
Recommended storage for kit components is 2 - 8°C.
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MILLIPLEX is a registered trademark of Merck KGaA, Darmstadt, Germany
xMAP is a registered trademark of Luminex Corp
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