Revvity, 64ATF4TPEH, HTRF Human Total ATF4 Detection Kit, 10,000 Assay Points

Overview
ATF4, a pivotal transcription factor in the EIF2a/ATF-4 signaling pathway, serves as the primary effector of the integrated stress response (ISR) triggered by PERK, HRI, GCN2, or PKR kinases. With a leucine Zipper domain (bZIP), ATF4 facilitates interactions with numerous proteins, acting as dimerization partners. Its multifaceted role extends across various tissues, significantly impacting obesity, glucose homeostasis, energy expenditure, and neural plasticity regulation.
As a stress-induced bZIP transcription factor, ATF4 binds to the cAMP response element, marking its involvement in the ISR, which encompasses responses to amino acid and glucose depletion, ER stress, and oxidative stress. This response entails the phosphorylation of eIF2alpha by various kinases, leading to global mRNA translation attenuation and ATF4 translation upregulation. Depending on post-translational modifications and binding partners, ATF4 can either promote pro-survival or apoptosis.
Additionally, ATF4's stability is subject to regulation through polyubiquitinylation, which mediates its proteasomal degradation.
How it works
Total ATF4 assay principle
The Total ATF4 assay quantifies the expression level of ATF4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total ATF4 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of ATF4 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 ATF4 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of HTRF Total ATF4 detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Total ATF4 one-plate assay protocol
Detection of Total ATF4 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 facilitates miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Buffer/Solvent-Lysis Buffer 4
Detection Modality-HTRF
Host Species-Human
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-ATF4
Target Class-Phosphoproteins
Technology-TR-FRET
Therapeutic Area-Neuroscience Oncology
Unit Size-10,000 assay points