Phospho-mTOR (Ser2448) cellular kit

The phospho-mTOR (Ser2448) kit enables the cell-based quantitative detection of phosphorylated mTOR.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The phospho-mTOR (Ser2448) kit enables the cell-based quantitative detection of phosphorylated mTOR.
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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.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • PRECISION

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 protein’s phosphorylation state under a no-wash assay format.
Phospho-mTor (Ser2448) assay principle

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) 2-plate assay protocol

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.
Phospho-mTor (Ser2448) 1-plate assay protocol

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.
HTRF assay compared to Western Blot using phospho-mTOR cellular assay

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.
Insulin dose-response on NIH 3T3 cells

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

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.
Phospho-mTor signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

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