Phospho-4EBP1 (Thr37/46) cellular kit
Highly sensitive 4EBP1 phosphorylation cellular assay kit
Phospho-EIF2 alpha (Ser52) cellular kit HTRF®
The phospho-EIF2 alpha (Ser52) kits are optimal for cell-based detection of Ser52 phosphorylation on EIF2 as a readout of cellular stress signaling pathways.
See more
-
Highly specific
-
Ease-of-use
Overview
The phospho-EIF2 alpha assay is designed for the cell-based quantitative detection of Ser52 phosphorylation on EIF2 alpha. In NASH, lipid accumulation in hepatocytes (steatosis) induces oxidative and ER stresses, resulting in an increase in 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.
Benefits
- COMPATIBLE WITH MANY CELL TYPES
- SPECIFICITY
Phospho-EIF2 alpha (Ser52) assay principle
The Phospho-EIF2 alpha (Ser52) assay measures EIF2 alpha when phosphorylated at Ser52. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-EIF2 alpha (Ser52) 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 proteins phosphorylation state under a no-wash assay format.
Phospho-EIF2 alpha (Ser52) 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-EIF2 alpha (Ser52) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-EIF2 alpha (Ser52) 1-plate assay protocol
Detection of Phosphorylated EIF2 alpha (Ser52) 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 Phospho-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. Results show the HTRF Phoshpo-EIF2a assay is 16 fold more sensitive than the Western Blot technique.
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).
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Open R&D: Sanofi Access Platform
In collaboration with Sanofi - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Webinar: NASH - Fibrosis Research and Drug Discovery
Featuring a panel of experts - Videos
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Product Insert EIF2a P-S52 Kit / 64EF2PEG-64EF2PEH
64EF2PEG-64EF2PEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (ELL) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (ITA) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (SPA) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) EIF2a P-S52 Kit / 64EF2PEG
64EF2PEG - Safety Data Sheet
Safety Data Sheet (DEU) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (ELL) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (ITA) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (SPA) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) EIF2a P-S52 Kit / 64EF2PEH
64EF2PEH - Safety Data Sheet
Safety Data Sheet (DEU) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (ELL) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (ITA) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (SPA) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Safety Data Sheet (ENG-US) EIF2a P-S52 Kit / 64EF2PEY
64EF2PEY - Safety Data Sheet
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
Let's find your reader
You might be interested in
Latest news