The total EIF2 alpha kit monitors cellular EIF2 alpha expression level and can be used as a normalization assay for the phospho-EIF2 alpha kit.
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The total EIF2 alpha kit monitors cellular EIF2 alpha expression level and can be used as a normalization assay for the phospho-EIF2 alpha kit.
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Overview

The Total EIF2 alpha cellular assay monitors total EIF2 alpha and is used as a normalization assay with the phospho-EIF2 alpha kit. In NASH, lipid accumulation in hepatocytes (steatosis) induces oxidative and ER stresses, resulting in an increase of Phospho-EIF2alpha (Ser52). Phosphorylated EIF2 alpha 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.

Total-EIF2 alpha assay principle

The Total-EIF2 alpha assay quantifies the expression level of EIF2 alpha in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-EIF2 alpha assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of EIF2 alpha in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-EIF2 alpha 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 Total EIF2 alpha HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-EIF2 alpha 1-plate assay protocol

Detection of total EIF2 alpha 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.

HTRF vs WB using total EIF2a assay 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 Total EIF2 alpha kit, only 0.2 µg of total proteins were sufficient for minimal signal detection, demonstrating that the HTRF assay is 4 fold more sensitive than the Western Blot.

Pharmaco effects of Febrifugine on Total and Phospho (Ser52) EIF2a

HepG2 and NIH-3T3 cells were treated with increasing concentrations of Febrifugine. After lysis, lysates were transferred 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.

Pharmaco effects of Thapsigargin on Total and Phospho (Ser52) EIF2a

Dose response experiments were performed on HepG2 cells with Thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+/ATPase by inducing ER stress. Using the two plate protocol, cells were treated with increasing concentrations of Thapsigargin and lysed. Lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-EIF2 alpha or total EIF2 alpha detection antibodies. Results showed EIF2 alpha expression level remains stable, demonstrating that there is no cytotoxic effect of the compound on cells.

Pharmaco effects of GSK2606414 on Total and Phospho (Ser52) EIF2a

HepG2 and C2C12 cells were cultured and treated with increasing concentrations of GSK2606414, a selective PERK inhibitor, before activation with 500 nM Thapsigargin. After medium removal, the cells were lysed 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.

Pharmaco effects of Palmitate on Total and Phospho (Ser52) EIF2a

Dose response experiments were performed on HepG2 cells with a solution of palmitate conjugated to BSA using the two plate assay protocol. Cells were then treated with increasing concentrations of palmitate/BSA for 6 hours at 37°C - 5% CO2. After lysis buffer, 16 µL of lysate were transferred twice over into a low volume white microplate before finally adding phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded and by way of a 2-way ANOVA test, results demonstrated Palmitate induces a good, significant increase in the phosphorylation of EIF2 alpha on SER52.

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).
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Product Insert EIF2a total Kit / 64NEFPEG-64NEFPEH

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