Phospho-4EBP1 (Thr37/46) Assay principle

The Phospho-4EBP1 (Thr37/46) assay measures 4EBP1 when phosphorylated at Thr37/46. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-4EBP1 (Thr37/46) 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-4EBP1 (Thr37/46) 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-4EBP1 (Thr37/46) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-4EBP1 (Thr37/46) 1-plate assay protocol

Detection of Phosphorylated 4EBP1 (Thr37/46) 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-4EBP1 HTRF assay performance compared to WB

Human HEK293 cells were grown in a T175 flask at 37°C until 80% confluency. After removal of cell culture medium, 3 mL of supplemented lysis buffer were added and incubated for 30 minutess. 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

Species compatibility: PP242 + Rapamycin in murine NIH3T3 cells

Murine NIH3T3 cells were incubated with various concentrations of tPP242 and Rapamycin, followed by stimulation with Insulin for 30 minutess at 37°C. After lysis, 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.

Translation is initiated when a ribosome is recruited to the 5 end of an mRNA. The eIF4E-binding proteins, such as 4EBP1, block translation by binding to eIF4E and preventing recruitment of the translation machinery. Hypophosphorylated 4EBP1 downregulates translation, whereas 4EBP1 hyperphosphorylation by mTOR 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.