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HTRF Human and Mouse Phospho-mTOR (Ser2448) Detection Kit, 500 Assay Points

The phospho-mTOR (Ser2448) kit enables the cell-based quantitative detection of phosphorylated mTOR.

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Feature	Specification
Application	細胞シグナル伝達
Sample Volume	16 µL

The phospho-mTOR (Ser2448) kit enables the cell-based quantitative detection of phosphorylated mTOR.

View product information

Product variants

Unit Size: 500 assay points

Part #:

64TORPEG

Unit Size: 10,000 assay points

Part #:

64TORPEH

Unit Size: 50,000 assay points

Part #:

64TORPEY

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

Overview

The Phospho-mTOR (Ser2448) cellular assay kit is ideal for measuring phosphorylated mTOR directly in cells. Using a streamlined protocol, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.

How it works

Phospho-mTor (Ser2448) assay principle

The Phospho-mTor (Ser2448) assay measures mTor when phosphorylated at Ser2448. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-mTor (Ser2448) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.

Phospho-mTor (Ser2448) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-mTor (Ser2448) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-mTor (Ser2448) 1-plate assay protocol

Detection of Phosphorylated mTor (Ser2448) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Assay validation

HTRF assay compared to WB using phospho-mTOR cellular assay

Hek293 cells were grown in a T175 flask 37°C, 5% Co2, 2 days.After elimination of cell culture medium, 3 mL 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.

Insulin dose-response on NIH 3T3 cells

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

Inhibition effect of Rapamycin 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 minutes lysis incubation time, inhibition of the basal level of phosphorylated mTOR was measured using the one-plate assay protocol.

Simplified pathway

Simplified Pathway for Phospho-mTor (Ser2448) assay kit

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 itself phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway, and autophosphorylated at Ser2481.

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Phosphorylation
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Cardiovascular Metabolism/Diabetes NASH/Fibrosis Neuroscience Oncology & Inflammation
Unit Size	500 assay points