Revvity, 64NEFPEG, HTRF Human and Mouse Total eIF2α Detection Kit, 500 Assay Points

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.
How it works
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 proteins 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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Total
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