Phospho-mTOR (Ser2448) Cellular Assay Kit

HTRF® cellular assay kit for measuring phosphorylated mTOR directly in cells

Based on our homogeneous and robust HTRF assays, the Phospho-mTOR kit is designed for detecting and studying activated mTOR when phosphorylated at Ser2448 directly in whole cells. Using a streamlined protocol, amenable to low-volume format, this kit can be used from basic research to High Throughput drug screening.

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Assay Principle

The assay can be run with cell lysates or using whole cells.

Upon activation, mTOR is phosphorylated on Ser2448 and after the lysis of the cell membrane, phospho-mTOR can be detected using the kit reagents. The assays are based on sandwich immunoassays, each involving two monoclonal antibodies: the anti-phospho-mTOR antibody labeled with Eu3+-cryptate and the anti-mTOR antibody labeled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.

The assays can be run under a two-plate protocol. They can also be further streamlined to a one-step assay.

Two-plate assay protocol

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated mTOR by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated mTOR with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, enables miniaturization while maintaining HTRF quality.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase  that functions as an ATP and amino acid sensor and plays  a key role in cell growth and homeostasis. As mTOR may be abnormally regulated in tumors, it is currrently under investigation as a potential target for anti-cancer therapy. mTOR is a common element of 2  signalling complexes: mTORC1 involved in nutrient, energy sensing, and  control of protein synthesis, and mTORC2 identified as the kinase responsible for AKT phosphorylation at Ser473. mTOR is itself phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway, and autophosphorylated at Ser2481.

Product Performance

1. Western Blot versus HTRF assay

Hek293 cells were grown in a T175 flask 37°C, 5% Co2, 2 days.After elimination of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 min.Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF.HTRF assay shows the same level of sensitivity as Western Blot: 1560 cells for the two technologies.

2. Insulin dose-response on NIH 3T3 cells

Murine NIH 3T3 cells (100,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of forskolin. After a 30 minute lysis incubation time, phosphorylated mTOR was measured using the two-plate assay protocol.

3. Inhibition effect of Rapamycin inhibitor on basal level of Jurkat cells

Jurkat cells (100,000 cells/well) were incubated for 30 min at 37°C with various concentrations of inhibitor. After a 30 minute lysis incubation time, inhibition of the basal level of phosphorylated mTOR was measured using the one-plate assay protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
mTOR phospho-S2448 kit 500 tests64TORPEG
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mTOR phospho-S2448 kit 10,000 tests64TORPEH
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mTOR phospho-S2448 kit 50,000 tests64TORPEY
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Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
mTOR phospho-S2448 kit control lysate64TORTDA
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Documents