Phospho-EIF2 alpha (Ser52) & Total EIF2 alpha Cellular Assay Kits

Total & Phospho-EIF2 α (Ser52) assays for monitoring stress pathway activation

These cell-based assays are designed to monitor the expression level and the phosphorylation of EIF2 alpha (on Ser52), which is a readout of the activation of stress signaling pathways.

In NASH, lipid accumulation in hepatocytes (steatosis) induces oxidative and ER stresses, resulting in the increase of Phospho-EIF2alpha (Ser52). Phosphorylated EIF2alpha reduces global protein synthesis and activates ATF4 (Activating Transcription Factor 4) which modulates the expression of specific genes involved in the regulation of metabolism, nutrient uptake, the redox status of cells and apoptosis.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total EIF2 alpha assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-EIF2 alpha antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to EIF2 alpha, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. 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 assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-EIF2 alpha can be quantitatively detected using the HTRF phospho-EIF2 alpha (Ser52) and total EIF2 alpha cellular assay 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, lysates are subsequently transferred to a 384-sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

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.

Product performances

1. Comparison of HTRF and Western Blot Phospho and total EIF2α assays on human HeLa cells

HeLa cells were grown in a T175 cm² flask for 2 days in MEM alpha medium supplemented with 10% FBS. The cells were treated for 10 minutes with 100 nM Calyculin A at 37°C before lysis with 3 mL of 1X supplemented lysis buffer #4. The cell lysate was serially diluted in the supplemented lysis buffer #4 and 16 µL of each dilution were analyzed in parallel by HTRF or by Western Blot.

Using the HTRF® Phospho-EIF2 alpha kit, just 0.1 µg of total proteins were sufficient for minimal signal detection, while 1.6 µg were needed for a Western Blot signal. The HTRF assay is therefore 16 fold more sensitive than the Western Blot technique. Using the HTRF® Total EIF2 alpha kit, only 0.2 µg of total proteins were sufficient for minimal signal detection while 0.8 µg were needed for a Western Blot signal, demonstrating that the HTRF assay is 4 fold more sensitive than the Western Blot.

2. Pharmacological effects of compounds on Total and Phospho (Ser52) EIF2 alpha

2.1 Febrifugine

Febrifugine, a prolyl tRNA synthase, is known to act on GCN2 kinase.

Dose response experiments were performed on the human hepatoma cell line HepG2 or on the mouse fibroblastic cell line NIH-3T3 with Febrifugine, using the two plate assay protocol for adherent cells. Cells were plated at 50K cells/well for 24h at 37°C - 5% CO2 in DMEM medium + 10 % FBS.

The cells were treated with increasing concentrations of Febrifugine for 30 minutes at 37°C - 5% CO2. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation.

Febrifugine treatment induces a good increase in EIF2 alpha phosphorylation on Ser52, while its expression level remains stable.

2.2 Thapsigargin

Thapsigargin is an inhibitor of the sarco/endoplasmic reticulum Ca2+/ATPase characterized as inducing ER stress.

Dose response experiments were performed on HepG2 cells with Thapsigargin, using the two plate assay protocol for adherent cells. Cells were plated at 50Kcells/well for 24h at 37°C - 5% CO2 in DMEM medium + 10 % FBS. The cells were treated with increasing concentrations of Thapsigargin for 30 minutes at 37°C - 5%CO2. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho-EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation.

Thapsigargin induces ER stress, leading to the activation of the kinase PERK which in turn phosphorylates EIF2 alpha on Ser52. The EIF2 alpha expression level remains stable, demonstrating that there is no cytotoxic effect of the compound on cells.

2.3 GSK2606414

GSK2606414 is a selective PERK inhibitor.

Dose response experiments were performed on the human hepatoma cell line HepG2 or on the mouse myoblast cell line C2C12 followed by activation with Thapsigargin, using the two plate assay protocol for adherent cells. Cells were plated at 50Kcells/well for 24h at 37°C - 5% CO2 in DMEM medium + 10 % FBS. The cells were then treated with increasing concentrations of GSK2606414 for 1 hour at 37°C before activation for 30 minutes with 500 nM Thapsigargin. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, the PERK inhibitor GSK2606414 induces a significant decrease in the phosphorylation of EIF2 alpha on Ser52. The compound has no effect on the expression level of EIF2 alpha, which remains stable.

2.4 Palmitate effects

Palmitate is one of the factors involved in ER stress pathway activation in metabolic disorders (NAFLDs).

Dose response experiments were performed on HepG2 cells with a solution of palmitate conjugated to BSA (for each dose the appropriate BSA control was performed) using the two plate assay protocol for adherent cells. Cells were plated at 50Kcells/well for 24h at 37°C - 5% CO2 in OPTI-MEM medium without FBS.

The cells were treated with increasing concentrations of palmitate/BSA for 6 hours at 37°C - 5% CO2. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation. The results presented below correspond to the signal of the phospho-protein normalized by the signal of the total protein. Biostatistics were performed using a two-way ANOVA test.

Palmitate induces a good, significant increase in the phosphorylation of EIF2 alpha on SER52.

Simplified pathway

Function and regulation of EIF2alpha

EIF2 (Eukaryotic Initiation Factor 2) is required in the initiation of protein translation by transferring the initiator methionyl tRNA to the 40S Ribosomal subunit of the ribosome. Phosphorylation of the EIF2 alpha subunit is a mechanism which downregulates global protein synthesis under a variety of cellular stress conditions, enabling cells to conserve resources, while a new gene expression program is adopted to prevent stress damage.

There are various cellular stresses, such as Endoplasmic Reticulum (ER) stress, amino acid deficiency, or oxidative stress. Four kinases can phosphorylate EIF2 alpha in response to a distinct type of stress(es): HRI (heme deprivation in erythroid cells), GCN2 (amino acid deficiency and nutrient deprivation), PKR (viral infection), and PERK (ER stress).

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
EIF2 alpha phospho-S52 kit - 500 tests64EF2PEG
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EIF2 alpha phospho-S52 kit - 10,000 tests64EF2PEH
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EIF2 alpha phospho-S52 kit - 50,000 tests64EF2PEY
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EIF2 alpha total kit - 500 tests64NEFPEG
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EIF2 alpha total kit - 10,000 tests64NEFPEH
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Companion products

DescriptionCat. noProduct insertMSDS
EIF2 alpha phospho-S52 kit control lysate64EF2TDA
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EIF2 alpha total kit control lysate64NEFTDA
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