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Microplates

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Major pharma and biotech companies have increasingly turned their attention to regulating the mTOR pathway in recent cancer drug research. Of particular note is mTOR’s relationship with p70 S6K kinase, as borne out by recent literature addressing this target.

For example, Busch, et. al., in Experimental Cell Biology
(doi:10.1016/j.yexcr.2009.01.026), found that:

“Rapamycin inhibited mTOR kinase activity as demonstrated with a lower phosphorylation level of the mTOR substrate p70 S6 kinase (S6K).… While tyrosine phosphorylation of STAT3 was unaffected by mTOR inhibition and knockdown, serine phosphorylation was diminished. We conclude that mTOR signaling is one major mechanism in a tightly regulated network of intracellular signal pathways including the JAK/STAT system to regulate invasion in human trophoblast cells by secretion of enzymes that remodel the extra-cellular matrix (ECM) such as MMP-2, -9, uPA and PAI-1.”

Further, as noted by Memmott and Dennis in Cellular Signalling (Volume 21, Issue 5, May 2009, Pages 656-664), the well-known role of mTOR and p70 S6K served as a starting point for their research in “Akt-dependent and -independent mechanisms of mTOR regulation in cancer.” They write:

“The serine-threonine kinase mTOR is a master regulator of protein synthesis, and plays important roles in other biological processes that support cell growth and survival, such as angiogenesis and autophagy. mTOR exists in two functionally distinct complexes in cells, namely mTORC1 and mTORC2. mTORC1 is composed of mTOR, Raptor, mLST8, and PRAS40, and is sensitive to inhibition by the macrolide antibiotic rapamycin. Importantly, mTORC1 activates S6K1 (p70 ribosomal protein S6 kinase) and inactivates 4E-BP1 (eIF4E binding protein 1), which promotes protein translation and cell growth.”

Your Cellular and Biochemical mTOR Kinase Connection

PerkinElmer offers two choices for measuring phosphorylation of p70 S6K: biochemical assay TR-FRET LANCE Ultra and cellular assays based on AlphaScreen for measuring phosphorylation of endogenous cellular p70 S6K.

Biochemical: Easily miniaturizable, demonstrated and validated performance for mTOR research

LANCE® Ultra is a proven platform from the company that first commercialized Europium-based TR-FRET, and which offers specialized labeling expertise for Europium-labeled antibodies.

LANCE Ultra assay:

• Spends less time in assay development performing optimization
• TR-FRET technology easily lends itself to your lab workflows
• Readily miniaturizable, for 96-, 384-, and 1536-well formats

PerkinElmer Offers New Products for p70 S6K Phosphorylation - LANCE Ultra Biochemical Assay and AlphaScreen SureFire Cellular Assay

LANCE Ultra p70 S6K Kinase Assay

LANCE Ultra TR-FRET assays shows excellent S/B and Z’ values.

Enzyme Inhibition Curve

Z’ Factor Determination

In addition to mTOR, the proprietary LANCE Ultra ULight p70 S6K assay has been validated for the following kinases:

• CDK6/CycD3
• COT
• HGK
• IKKα
• IKKε
• IRAK1
• IRAK4
• MAP4K2
• MINK

• MST1
• NEK1
• NEK2
• NEK6
• NEK7
• PEK
• PLK1
• TAOK2

Click here to access detailed protocol information.

AlphaScreen SureFire p70 S6K Assay for Detection of Endogenous Substrate

Tests performed on multiple cell types shows positive response to the known mTOR inhibitor, rampamycin. Phosphorylation of p70 S6K at Thr389 proves to be a good marker in several cell types and is dependent on expression levels in those cells.

MCF7 cells

MCF7 cells harvested from 70% confluent flasks were plated into 96-well microplates at a density of 40,000 cells/well in media containing 10% FBS. The following day, the cells were treated for 2 hours with various concentrations of rapamycin, diluted in the same media, after which the cells were stimulated with 10 μg/mL insulin for 30 min at room temp. The media was removed from the wells, and the cells were lysed with 50 μL of freshly-prepared 1X Lysis buffer with gentle shaking for 10 min. A small portion of lysate (4 μL) was transferred to a low-volume 384-well proxiplate, and analyzed for p70S6K phosphorylation at Thr389 using the standard AlphaScreen SureFire protocol.

Primary fibroblast cells isolated from a 26 y/o donor and used at passage 5, were plated into 96-well microplates at a density of 40,000 cells/well in media containing 10% FBS. The following day, the cells were treated for 2 hours with various concentrations of rapamycin, diluted in the same media, after which the cells were stimulated with 50 ng/mL PDGF for 30 min at 37°C. The media was removed from the wells, and the cells were lysed with 50 μL of freshly-prepared 1X Lysis buffer with gentle shaking for 10 min. A small portion of lysate (4μL) was transferred to a low-volume 384-well proxiplate, and analyzed for p70S6K phosphorylation at Thr389 using the standard AlphaScreen SureFire protocol.

Map your pathway with more AlphaScreen SureFire mTOR signaling pathway kits:

• PDK-1
• AKT
• BAD
• GSK
• mTOR
• p70S6K
• 4E-BP1
• S6RP
• Caspase 9

Visit www.perkinelmer.com/kinaseconnection to learn more about all of our Cellular and Biochemical assay platforms.

©2009 PerkinElmer, Inc. All rights reserved.

LANCE and AlphaScreen are registered trademarks of PerkinElmer, Inc. SureFire is a registered trademark of TGR Biosceince PTY, LTD. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries are the property of their respective owners.