Phospho-EIF4E (Ser209) Cellular Assay Kit

HTRF® cellular assay kit for measuring endogenous phospho-eIF4E protein directly in cells.

Based on our TR-FRET homogeneous and robust HTRF® technology, the phospho- eIF4E assay kit is designed for the detection and analysis of dose-response inhibition and activation of endogenous eIF4E, phosphorylated at Ser209, directly in your cell type of interest. Using a streamlined mix-and-read no-wash protocol, this kit is amenable for any use from basic research to all drug screening phases.

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

The phospho- eIF4E assay kit can be run with cell lysates or whole cells.

Upon activation, eukaryotic translation initiation factor 4E (eIF4E) is phosphorylated on Ser209. After cell lysis, phospho- eIF4E can be detected and studied using the HTRF® kit reagents. This sandwich immunoassay involves two monoclonal antibodies: an anti-phospho- eIF4E antibody labelled with Eu3+ cryptate and an anti- eIF4E antibody labelled with d2. These antibodies may be pre-mixed and added in a single dispensing step for direct detection.

The assays can be run under a two-plate protocol, or 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 eIF4E by HTRF® reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated eIF4E with HTRF® reagents is performed in a single plate used for plating, stimulation, lysis and detection. No washing steps are required. This protocol, designed for HTS, enables 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

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

EIF4E, eukaryotic translation initiation factor 4E, initiates eukaryotic protein synthesis by binding to the 5’ end of the mRNA cap structure and directing its association with the ribosomes. EIF4E enables the expression of key genes such as cyclins, c-myc, and vascular endothelial growth factor, involved in cell proliferation, angiogenesis and cell survival. EIF4E combines with eIF4A, eIF3 and eIF4G to form the eIF4F complex, which loads mRNAs onto the 40S ribosomal subunit. The activity of eIF4E is regulated at two levels: it is activated by phosphorylation induced by MAPK/ MNK1 and by the release of an inhibitory complex with 4EBP1.The latter is controlled by the mTOR pathway, which liberates eIF4E from this inhibitory interaction by phosphorylation, allowing the initiation of translation. Overexpression of phosphorylated eIF4E at Ser209 is associated with tumorgenesis, metastasis, and poor overall survival in several cancers, e.g. breast, colon, lung, prostate, breast, and lymphoid.

Product Performance

1. Western Blot versus HTRF assay

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2, until 80% confluency for 1 day. Day 2: After removal of cell culture medium, 3ml of supplemented lysis buffer were added and the sample was incubated for 30 min. Soluble supernatants were collected after 10 min of centrifugation. Equal amounts of lysates in serial dilutions were analyzed in a side by side comparison of Western Blot and HTRF®. 16 µl of serial dilutions were analyzed in parallel by HTRF® and Western Blot. Note that 80,000 cells correspond roughly to 60 µg of proteins.328 cells can be detected by using HTRF® phospho-eIF4E (Ser209) whereas 5250 cells are needed for the Western Blot. The HTRF® assay is 8-fold more sensitive than Western Blot.

2. Dose-response of CGP57380 on NIH3T3 cells

Murine NIH3T3 cells (50,000 cells/well) were incubated for 3 hours with various concentrations of CGP57380 inhibitor. After a 30 minute lysis incubation, inhibition of eIF4E phosphorylation was measured using the HTRF® phospho-eIF4E (Ser209) assay with the two-plate protocol.

3. Dose-response of CGP57380 on stimulated HeLa cells

Human HeLa cells (100,000 cells/well) were incubated with various concentrations of CGP57380 inhibitor, either for 30 minutes or 3 hours, followed by stimulation with 10 µM Anisomycin for 30 minutes at 37°C. After a 30 minute lysis incubation, inhibition of eIF4E phosphorylation was measured using the HTRF® phospho-eIF4E (Ser209) assay with the two-plate protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
EIF4E phospho-S209 kit - 500 tests64EF4PEG
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EIF4E phospho-S209 kit - 10,000 tests64EF4PEH
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EIF4E phospho-S209 kit - 50,000 tests64EF4PEY
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Companion products

DescriptionCat. noProduct insertMSDS
EIF4E phospho-S209 kit - 500 tests64EF4PEG
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EIF4E phospho-S209 kit control lysate64EF4TDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
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Phospho-total protein blocking reagent - 6 ml64KB1AAD
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Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
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Documents