Phospho-4EBP1 (Thr37/46) Cellular Assay Kit

Homogenous cell-based HTRF® assay kit for measuring endogenous phospho-4EBP1 as an mTOR read-out in the translational control pathway

Cisbio’s homogeneous and robust HTRF® phospho-4EBP1 assay kit is designed for the cellular detection and quantification of endogenous 4EBP1 protein, phosphorylated on Thr37/46. Based on the TR-FRET technology, the HTRF® phospho-4EBP1 sandwich immunoassay provides an ideal read-out solution, in any cell type, for mTOR signaling and the translational control pathway. With an optimized and streamlined mix-and-read, no-wash protocol, the phospho-4EBP1 ready-to-use kit contains all the reagents you need. By enabling all throughputs, HTRF® phospho-4EBP1 facilitates drug screening, mechanistic signaling pathway studies, analysis of compound MoA and target research by delivering enhanced convenience and more relevant, reliable results. Let your research flow faster and more efficiently with Cisbio’s smart phospho-protein assays!

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

HTRF® - the homogenous sandwich immunoassay

The phospho-4EBP1 assay is based on a TR-FRET sandwich immunoassay format involving two different specific antibody conjugates: an anti-phospho-4EBP1 antibody labeled with Eu3+-cryptate (donor) and an anti-total-4EBP1 antibody labeled with d2 (acceptor). Upon m-TOR signal transduction, 4EBP1 is phosphorylated on Thr37/46. After phosphorylation, the anti-phospho-4EBP1 antibody will recognize the phosphorylated residue, and the proximity of donor and acceptor will consequently lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for 384-well plate format but can easily be further miniaturized or upscaled. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The phospho-4EBP1 assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-4EBP1 can be quantitatively detected using the HTRF® phospho-4EBP1 kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection of phosphorylated 4EBP1, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This enables the cells' viability and confluence to be monitored in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No washing steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling miniaturization while maintaining a high quality output.

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

4EBP1 cell signaling

Translation is initiated when a ribosome is recruited to the 5’ end of an mRNA. The eIF4E-binding proteins, amongst others 4EBP1, prevent translation initiation by binding to the translation factor eIF4E, and precluding recruitment of the translation machinery to the mRNA. Hypophosphorylated 4EBP1 interacts with eIF4E to downregulate translation, whereas 4EBP1 hyperphosphorylation by mTOR, induced by cell stimulation with hormones, cytokines, or growth factors, abrogates eIF4E-binding activity and therefore promotes translation.
The assessment of cellular phospho-4EBP1 is an important tool to generally measure mTOR potency and to determine the activity of mTOR signaling in the presence of all the associated proteins that constitute the mTOR complexes.

Product Performance

HTRF® assay compared to Western Blot

Human HEK293 cells were grown in a T175 flask at 37°C until 80% confluency. After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 minutes. Soluble supernatants were collected after a 10 minute centrifugation. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.

Using HTRF® phospho-4EBP1 (THR37/46), just 400 cells are sufficient for minimal signal detection while 11,300 cells are needed for a Western Blot signal. The HTRF® assay is 32-fold more sensitive than the Western Blot.

Pharmacological response on phospho-4EBP1 as read-out for inhibition of mTORC1 activity

Jurkat, A431 and SW480 cells were seeded at 25,000 cells per well in a 96-well plate with DMEM+10%FCS for 24 hours. The cells were treated for 3 hours with known pan-mTOR inhibitors, either with increasing concentrations of PP242 or BEZ235. The two-plate adherent protocol was applied to A431 and SW480 cells, whereas the two-plate suspension protocol was followed for Jurkat cells.

Species compatibility: PP242 and Rapamycin dose response in murine NIH3T3 cells

Murine NIH3T3 cells were incubated for 3 hours with various concentrations of the ATP competitor PP242 and the allosteric inhibitor Rapamycin, followed by stimulation with 1.0 µM of Insulin for 30 minutes at 37°C. After a 30 minute lysis incubation, inhibition of 4EBP1 phosphorylation was measured using the HTRF® phospho-4EBP1 (THR37/46) assay according to the two-plate protocol. PP242 is a potent pan-mTOR inhibitor of both mTORC1 (including Rapamycin-resistant 4EBP phosphorylation) and mTORC2, whereas Rapamycin treatment is known to have only partial or no effect on 4EBP1 phosphorylation. - Feldman et al. (2009) Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 7(2)

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
4EBP1 phospho-T37/46 kit - 500 tests64BP1PEG
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4EBP1 phospho-T37/46 kit - 10,000 tests64BP1PEH
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4EBP1 phospho-T37/46 kit - 50,000 tests64BP1PEY
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Companion products

DescriptionCat. noProduct insertMSDS
4EBP1 phospho-T37/46 kit control lysate64BP1TDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF

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