Revvity, 64ATG14TPEG, HTRF Human Total ATG14 Detection Kit, 500 Assay Points

Overview
ATG14, Autophagy related protein 14, or BAKOR for Beclin 1-associated autophagy-related key regulator, is a key player in the autophagosome nucleation step in macroautophagy. Upon cellular stress, the nutrient/energy-sensitive sensors mTOR and AMPK lead to the activation of the ULK1 complex which allows phosphorylation of ATG14 on Serine 29, in turn enabling the downstream activation of the PIK3C3 autophagosome nucleation complex.
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
Total ATG14 assay principle
The Total-ATG14 assay quantifies the expression level of ATG14 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total-ATG14 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 ATG14 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-ATG14 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total-ATG14 detection reagents. This protocol enables the cells' viability and confluence to be monitored

 

Total-ATG14 one-plate assay protocol
Detection of Total ATG14 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
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-500 assay points