Revvity, 64EF2PEG, HTRF Human and Mouse Phospho-eIF2α (Ser52) Detection Kit, 500 Assay Points

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.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Metabolism/Diabetes NASH/Fibrosis
Unit Size-500 assay points